Streptomyces avermitilis gene directing the ratio of B2:B1 avermectins

ABSTRACT

The present invention relates to polynucleotide molecules comprising nucleotide sequences encoding an aveC gene product, which polynucleotide molecules can be used to alter the ratio or amount of class 2:1 avermectins produced in fermentation cultures of  S. avermitilis.  The present invention further relates to vectors, host cells, and mutant strains of  S. avermitilis  in which the aveC gene has been inactivated, or mutated so as to change the ratio or amount of class 2:1 avermectins produced.

[0001] This application is a continuation-in-part of PCT/IB99/00130,filed Jan. 25, 1999, which claims priority from U.S. provisionalapplication 60/074,636, filed Feb. 13, 1998.

1. FIELD OF THE INVENTION

[0002] The present invention is directed to compositions and methods forproducing avermectins, and is primarily in the field of animal health.More particularly, the present invention relates to polynucleotidemolecules comprising nucleotide sequences encoding an AveC gene product,which can be used to modulate the ratio of class 2:1 avermectinsproduced by fermentation of cultures of Streptomyces avermitilis, and tocompositions and methods for screening for such polynucleotidemolecules. The present invention further relates to vectors, transformedhost cells, and novel mutant strains of S. avermitilis in which the aveCgene has been mutated so as to modulate the ratio of class 2:1avermectins produced.

2. BACKGROUND OF THE INVENTION 2.1. Avermectins

[0003] Streptomyces species produce a wide variety of secondarymetabolites, including the avermectins, which comprise a series of eightrelated sixteen-membered macrocyclic lactones having potent anthelminticand insecticidal activity. The eight distinct but closely relatedcompounds are referred to as A1 a, A1 b, A2 a, A2 b, B1 a, B1 b, B2 aand B2 b. The “a” series of compounds refers to the natural avermectinwhere the substituent at the C25 position is (S)-sec-butyl, and the “b”series refers to those compounds where the substituent at the C25position is isopropyl. The designations “A” and “B” refer to avermectinswhere the substituent at the C5 position is methoxy and hydroxy,respectively. The numeral “1” refers to avermectins where a double bondis present at the C22,23 position, and the numeral “2” refers toavermectins having a hydrogen at the C22 position and a hydroxy at theC23 position. Among the related avermectins, the B1 type of avermectinis recognized as having the most effective antiparasitic and pesticidalactivity, and is therefore the most commercially desirable avermectin.

[0004] The avermectins and their production by aerobic fermentation ofstrains of S. avermitilis are described in U.S. Pat. Nos. 4,310,519 and4,429,042. The biosynthesis of natural avermectins is believed to beinitiated endogenously from the CoA thioester analogs of isobutyric acidand S-(+)-2-methyl butyric acid.

[0005] A combination of both strain improvement through randommutagenesis and the use of exogenously supplied fatty acids has led tothe efficient production of avermectin analogs. Mutants of S.avermitilis that are deficient in branched-chain 2-oxo aciddehydrogenase (bkd deficient mutants) can only produce avermectins whenfermentations are supplemented with fatty acids. Screening and isolationof mutants deficient in branched-chain dehydrogenase activity (e.g., S.avermitilis, ATCC 53567) are described in European Patent (EP) 276103.Fermentaton of such mutants in the presence of exogenously suppliedfatty acids results in production of only the four avermectinscorresponding to the fatty acid employed. Thus, supplementingfermentations of S. avermitilis (ATCC 53567) with S-(+)-2-methylbutyricacid results in production of the natural avermectins A1 a, A2 a, B1 aand B2 a; supplementing fermentations with isobutyric acid results inproduction of the natural avermectins A1 b, A2 b, B1 b, and B2 b; andsupplementing fermentations with cyclopentanecarboxylic acid results inthe production of the four novel cyclopentylavermectins A1, A2, B1, andB2.

[0006] If supplemented with other fatty acids, novel avermectins areproduced. By screening over 800 potential precursors, more than 60 othernovel avermectins have been identified. (See, e.g., Dutton et at., 1991,J. Antibiot. 44:357-365; and Banks et al., 1994, Roy. Soc. Chem.147:16-26). In addition, mutants of S. avermitilis deficient in5-O-methyltransferase activity produce essentially only the B analogavermectins. Consequently, S. avermitilis mutants lacking bothbranched-chain 2-oxo acid dehydrogenase and 5-O-methyltransferaseactivity produce only the B avermectins corresponding to the fatty acidemployed to supplement the fermentation. Thus, supplementing such doublemutants with S-(+)-2-methylbutyric acid results in production of onlythe natural avermectins B1 a and B2 a, while supplementing withisobutyric acid or cyclopentanecarboxylic acid results in production ofthe natural avermectins B1 b and B2 b or the novel cyclopentyl B1 and B2avermectins, respectively. Supplementation of the double mutant strainwith cyclohexane carboxylic acid is a preferred method for producing thecommercially important novel avermectin, cyclohexylavermectin B1(doramectin). The isolation and characteristics of such double mutants,e.g., S. avermitilis (ATCC 53692), is described in EP 276103.

2.2. Genes Involved In Avermectin Biosynthesis

[0007] In many cases, genes involved in production of secondarymetabolites and genes encoding a particular antibiotic are foundclustered together on the chromosome. Such is the case, e.g., with theStreptomyces polyketide synthase gene cluster (PKS) (see Hopwood andSherman, 1990, Ann. Rev. Genet. 24:3766). Thus, one strategy for cloninggenes in a biosynthetic pathway has been to isolate a drug resistancegene and then test adjacent regions of the chromosome for other genesrelated to the biosynthesis of that particular antibiotic. Anotherstrategy for cloning genes involved in the biosynthesis of importantmetabolites has been complementation of mutants. For example, portionsof a DNA library from an organism capable of producing a particularmetabolite are introduced into a non-producing mutant and transformantsscreened for production of the metabolite. Additionally, hybridizationof a library using probes derived from other Streptomyces species hasbeen used to identify and clone genes in biosynthetic pathways.

[0008] Genes involved in avermectin biosynthesis (ave genes), like thegenes required for biosynthesis of other Streptomyces secondarymetabolites (e.g., PKS), are found clustered on the chromosome. A numberof ave genes have been successfully cloned using vectors to complementS. avermitilis mutants blocked in avermectin biosynthesis. The cloningof such genes is described in U.S. Pat. No. 5,252,474. In addition,Ikeda et al., 1995, J. Antibiot. 48:532-534, describes the localizationof a chromosomal region involving the C22,23 dehydration step (aveC) toa 4.82 Kb BamHI fragment of S. avermitilis, as well as mutations in theaveC gene that result in the production of a single component B2 aproducer. Since ivermectin, a potent anthelmintic compound, can beproduced chemically from avermectin B2 a, such a single componentproducer of avermectin B2 a is considered particularly useful forcommercial production of ivermectin.

[0009] Identification of mutations in the aveC gene that minimize thecomplexity of avermectin production, such as, e.g., mutations thatdecrease the B2:B1 ratio of avermectins, would simplify production andpurification of commercially important avermectins.

3. SUMMARY OF THE INVENTION

[0010] The present invention provides an isolated polynucleotidemolecule comprising the complete aveC ORF of S. avermitilis or asubstantial portion thereof, which isolated polynucleotide moleculelacks the next complete ORF that is located downstream from the aveC ORFin situ in the S. avermitilis chromosome. The isolated polynucleotidemolecule of the present invention preferably comprises a nucleotidesequence that is the same as the S. avermitilis AveC geneproduct-encoding sequence of plasmid pSE186 (ATCC 209604), or that isthe same as the nucleotide sequence of the aveC ORF of FIG. 1 (SEQ IDNO:1), or substantial portion thereof. The present invention furtherprovides an isolated polynucleotide molecule comprising the nucleotidesequence of SEQ ID NO:1 or a degenerate variant thereof.

[0011] The present invention further provides an isolated polynucleotidemolecule having a nucleotide sequence that is homologous to the S.avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC209604), or to the nucleotide sequence of the aveC ORF presented in FIG.1 (SEQ ID NO:1) or substantial portion thereof.

[0012] The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that encodes a polypeptidehaving an amino acid sequence that is homologous to the amino acidsequence encoded by the AveC gene product-encoding sequence of plasmidpSE186 (ATCC 209604), or the amino acid sequence of FIG. 1 (SEQ ID NO:2)or substantial portion thereof.

[0013] The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence encoding an AveC homolog geneproduct. In a preferred embodiment, the isolated polynucleotide moleculecomprises a nucleotide sequence encoding the AveC homolog gene productfrom S. hygroscopicus, which homolog gene product comprises the aminoacid sequence of SEQ ID NO:4 or a substantial portion thereof. In apreferred embodiment, the isolated polynucleotide molecule of thepresent invention that encodes the S. hygroscopicus AveC homolog geneproduct comprises the nucleotide sequence of SEQ ID NO:3 or asubstantial portion thereof.

[0014] The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that is homologous to the S.hygroscopicus nucleotide sequence of SEQ ID NO:3. The present inventionfurther provides an isolated polynucleotide molecule comprising anucleotide sequence that encodes a polypeptide that is homologous to theS. hygroscopicus AveC homolog gene product having the amino acidsequence of SEQ ID NO:4.

[0015] The present invention further provides oligonucleotides thathybridize to a polynucleotide molecule having the nucleotide sequence ofFIG. 1 (SEQ ID NO:1) or SEQ ID NO:3, or to a polynucleotide moleculehaving a nucleotide sequence which is the complement of the nucleotidesequence of FIG. 1 (SEQ ID NO:1) or SEQ ID NO:3.

[0016] The present invention further provides recombinant cloningvectors and expression vectors that are useful in cloning or expressinga polynucleotide of the present invention including polynucleotidemolecules comprising the aveC ORF of S. avermitilis or an aveC homologORF. In a non-limiting embodiment, the present invention providesplasmid pSE186 (ATCC 209604), which comprises the entire ORF of the aveCgene of S. avermitilis. The present invention further providestransformed host cells comprising a polynucleotide molecule orrecombinant vector of the invention, and novel strains or cell linesderived therefrom.

[0017] The present invention further provides a recombinanty expressedAveC gene product or AveC homolog gene product, or a substantial portionthereof, that has been substantially purified or isolated, as well ashomologs thereof. The present invention further provides a method forproducing a recombinant AveC gene product, comprising culturing a hostcell transformed with a recombinant expression vector, said recombinantexpression vector comprising a polynucleotide molecule having anucleotide sequence encoding an AveC gene product or AveC homolog geneproduct, which polynucleotide molecule is in operative association withone or more regulatory elements that control expression of thepolynucleotide molecule in the host cell, under conditions conducive tothe production of the recombinant AveC gene product or AveC homolog geneproduct, and recovering the AveC gene product or AveC homolog geneproduct from the cell culture.

[0018] The present invention further provides a polynucleotide moleculecomprising a nucleotide sequence that is otherwise the same as the S.avermitilis AveC allele, or the AveC gene product-encoding sequence ofplasmid pSE186 (ATCC 209604) or a degenerate variant thereof, or thenucleotide sequence of the aveC ORF of S. avermitilis as presented inFIG. 1 (SEQ ID NO:1) or a degenerate variant thereof, but that furthercomprises one or more mutations, so that cells of S. avermitilis strainATCC 53692 in which the wild-type aveC allele has been inactivated andthat express the polynucleotide molecule comprising the mutatednucleotide sequence produce a different ratio or amount of avermectinsthan are produced by cells of S. avermitilis strain ATCC 53692 thatinstead express only the wild-type aveC allele. According to the presentinvention, such polynucleotide molecules can be used to produce novelstrains of S. avermitilis that exhibit a detectable change in avermectinproduction compared to the same strain that instead expresses only thewild-type aveC allele. In a preferred embodiment, such polynucleotidemolecules are useful to produce novel strains of S. avermitilis thatproduce avermectins in a reduced class 2:1 ratio compared to that fromthe same strain that instead expresses only the wild-type aveC allele.In a further preferred embodiment, such polynucleotide molecules areuseful to produce novel strains of S. avermitilis that produce increasedlevels of avermectins compared to the same strain that instead expressesonly a single wild-type aveC allele. In a further preferred embodiment,such polynucleotide molecules are useful to produce novel strains of S.avermitilis in which the aveC gene has been inactivated.

[0019] The present invention provides methods for identifying mutationsof the aveC ORF of S. avermitilis capable of altering the ratio and/oramount of avermectins produced. In a preferred embodiment, the presentinvention provides a method for identifying mutations of the aveC ORFcapable of altering the class 2:1 ratio of avermectins produced,comprising:

[0020] (a) determining the class 2:1 ratio of avermectins produced bycells of a strain of S. avermitilis in which the aveC allele nativethereto has been inactivated, and into which a polynucleotide moleculecomprising a nucleotide sequence encoding a mutated AveC gene producthas been introduced and is being expressed;

[0021] (b) determining the class 2:1 ratio of avermectins produced bycells of the same strain of S. avermitilis as in step (a) but whichinstead express only the wild-type aveC allele or the ORF of FIG. 1 (SEQID NO:1) or a nucleotide sequence that is homologous thereto; and

[0022] (c) comparing the class 2:1 ratio of avermectins produced by theS. avermitilis cells of step (a) to the class 2:1 ratio of avermectinsproduced by the S. avermitilis cells of step (b); such that if the class2:1 ratio of avermectins produced by the S. avermitilis cells of step(a) is different from the class 2:1 ratio of avermectins produced by theS. avermitilis cells of step (b), then a mutation of the aveC ORFcapable of altering the class 2:1 ratio of avermectins has beenidentified. In a preferred embodiment, the class 2:1 ratio ofavermectins is reduced by the mutation.

[0023] In a further preferred embodiment, the present invention providesa method for identifying mutations of the aveC ORF or genetic constructscomprising the aveC ORF capable of altering the amount of avermectinsproduced, comprising:

[0024] (a) determining the amount of avermectins produced by cells of astrain of S. avermitilis in which the aveC allele native thereto hasbeen inactivated, and into which a polynucleotide molecule comprising anucleotide sequence encoding a mutated AveC gene product or comprising agenetic construct comprising a nucleotide sequence encoding an AveC geneproduct has been introduced and is being expressed;

[0025] (b) determining the amount of avermectins produced by cells ofthe same strain of S. avermitilis as in step (a) but which insteadexpress only a single aveC allele having the nucleotide sequence of theORF of FIG. 1 (SEQ ID NO:1) or a nucleotide sequence that is homologousthereto; and

[0026] (c) comparing the amount of avermectins produced by the S.avermitilis cells of step (a) to the amount of avermectins produced bythe S. avermitilis cells of step (b); such that if the amount ofavermectins produced by the S. avermitilis cells of step (a) isdifferent from the amount of avermectins produced by the S. avermitiliscells of step (b), then a mutation of the aveC ORF or a geneticconstruct capable of altering the amount of avermectins has beenidentified. In a preferred embodiment, the amount of avermectinsproduced is increased by the mutation.

[0027] The present invention further provides recombinant vectors thatare useful for making novel strains of S. avermitilis having alteredavermectin production. For example, the present invention providesvectors that can be used to target any of the polynucleotide moleculescomprising the mutated nucleotide sequences of the present invention tothe site of the aveC gene of the S. avermitilis chromosome to eitherinsert into or replace the aveC allele or ORF or a portion thereof byhomologous recombination. According to the present invention, however, apolynucleotide molecule comprising a mutated nucleotide sequence of thepresent invention provided herewith can also function to modulateavermectin biosynthesis when inserted into the S. avermitilis chromosomeat a site other than at the aveC gene, or when maintained episomally inS. avermitilis cells. Thus, the present invention also provides vectorscomprising a polynucleotide molecule comprising a mutated nucleotidesequence of the present invention, which vectors can be used to insertthe polynucleotide molecule at a site in the S. avermitilis chromosomeother than at the aveC gene, or to be maintained episomally. In apreferred embodiment, the present invention provides gene replacementvectors that can be used to insert a mutated aveC allele into the S.avermitilis chromosome to generate novel strains of cells that produceavermectins in a reduced class 2:1 ratio compared to the cells of thesame strain which instead express only the wild-type aveC allele.

[0028] The present invention further provides methods for making novelstrains of S. avermitilis comprising cells that express a mutated avecallele and that produce altered ratios and/or amounts of avermectinscompared to cells of the same strain of S. avermitilis that insteadexpress only the wild-type avec allele. In a preferred embodiment, thepresent invention provides a method for making novel strains of S.avermitilis comprising cells that express a mutated aveC allele and thatproduce an altered class 2:1 ratio of avermectins compared to cells ofthe same strain of S. avermitilis that instead express only a wild-typeaveC allele, comprising transforming cells of a strain of S. avermitiliswith a vector that carries a mutated aveC allele that encodes a geneproduct that alters the class 2:1 ratio of avermectins produced by cellsof a strain of S. avermitilis expressing the mutated allele compared tocells of the same strain that instead express only the wild-type aveCallele, and selecting transformed cells that produce avermectins in analtered class 2:1 ratio compared to the class 2:1 ratio produced bycells of the strain that instead express the wild-type aveC allele. In apreferred embodiment, the class 2:1 ratio of avermectins produced isreduced in cells of the novel strain.

[0029] In a further preferred embodiment, the present invention providesa method for making novel strains of S. avermitilis comprising cellsthat produce altered amounts of avermectin, comprising transformingcells of a strain of S. avermitilis with a vector that carries a mutatedaveC allele or a genetic construct comprising the aveC allele, theexpression of which results in an altered amount of avermectins producedby cells of a strain of S. avermitilis expressing the mutated aveCallele or genetic construct as compared to cells of the same strain thatinstead express only the wild-type aveC allele, and selectingtransformed cells that produce avermectins in an altered amount comparedto the amount of avermectins produced by cells of the strain thatinstead express only the wild-type aveC allele. In a preferredembodiment, the amount of avermectins produced is increased in cells ofthe novel strain.

[0030] In a further preferred embodiment, the present invention providesa method for making novel strains of S. avermitilis, the cells of whichcomprise an inactivated aveC allele, comprising transforming cells of astrain of S. avermitilis that express any aveC allele with a vector thatinactivates the aveC allele, and selecting transformed cells in whichthe aveC allele has been inactivated.

[0031] The present invention further provides novel strains of S.avermitilis comprising cells that have been transformed with any of thepolynucleotide molecules or vectors comprising a mutated nucleotidesequence of the present invention. In a preferred embodiment, thepresent invention provides novel strains of S. avermitilis comprisingcells which express a mutated aveC allele in place of, or in additionto, the wild-type aveC allele, wherein the cells of the novel strainproduce avermectins in an altered class 2:1 ratio compared to cells ofthe same strain that instead express only the wild-type aveC allele. Ina more preferred embodiment, the cells of the novel strain produceavermectins in a reduced class 2:1 ratio compared to cells of the samestrain that instead express only the wild-type aveC allele. Such novelstrains are useful in the large-scale production of commerciallydesirable avermectins such as doramectin.

[0032] In a further preferred embodiment, the present invention providesnovel strains of S. avermitilis comprising cells which express a mutatedaveC allele, or a genetic construct comprising the aveC allele, in placeof, or in addition to, the aveC allele native thereto, which results inthe production by the cells of an altered amount of avermectins comparedto the amount of avermectins produced by cells of the same strain thatinstead express only the wild-type aveC allele. In a preferredembodiment, the novel cells produce an increased amount of avermectins.

[0033] In a further preferred embodiment, the present invention providesnovel strains of S. avermitilis comprising cells in which the aveC genehas been inactivated. Such strains are useful both for the differentspectrum of avermectins that they produce compared to the wild-typestrain, and in complementation screening assays as described herein, todetermine whether targeted or random mutagenesis of the aveC geneaffects avermectin production.

[0034] The present invention further provides a process for producingavermectins, comprising culturing cells of a strain of S. avermitilis,which cells express a mutated aveC allele that encodes a gene productthat alters the class 2:1 ratio of avermectins produced by cells of astrain of S. avermitilis expressing the mutated aveC allele compared tocells of the same strain which do not express the mutated aveC allelebut instead express only the wild-type aveC allele, in culture mediaunder conditions that permit or induce the production of avermectinstherefrom, and recovering said avermectins from the culture. In apreferred embodiment, the class 2:1 ratio of avermectins produced bycells expressing the mutation is reduced. This process providesincreased efficiency in the production of commercially valuableavermectins such as doramectin.

[0035] The present invention further provides a process for producingavermectins, comprising culturing cells of a strain of S. avermitilis,which cells express a mutated aveC allele or a genetic constructcomprising an aveC allele that results in the production of an alteredamount of avermectins produced by cells of a strain of S. avermitilisexpressing the mutated aveC allele or genetic construct compared tocells of the same strain which do not express the mutated aveC allele orgenetic construct but instead express only the wild-type aveC allele, inculture media under conditions that permit or induce the production ofavermectins therefrom, and recovering said avermectins from the culture.In a preferred embodiment, the amount of avermectins produced by cellsexpressing the mutation or genetic construct is increased.

[0036] The present invention further provides a novel composition ofavermectins produced by a strain of S. avermitilis expressing a mutatedaveC allele of the present invention, wherein the avermectins areproduced in a reduced class 2:1 ratio as compared to the class 2:1 ratioof avermectins produced by cells of the same strain of S. avermitilisthat do not express the mutated avec allele but instead express only thewild-type aveC allele. The novel avermectin composition can be presentas produced in fermentation culture fluid, or can be harvestedtherefrom, and can be partially or substantially purified therefrom.

4. BRIEF DESCRIPTION OF THE FIGURES

[0037]FIG. 1. DNA sequence (SEQ ID NO:1) comprising the S. avermitilisaveC ORF, and deduced amino acid sequence (SEQ ID NO:2).

[0038]FIG. 2. Plasmid vector pSE186 (ATCC 209604) comprising the entireORF of the aveC gene of S. avermitilis.

[0039]FIG. 3. Gene replacement vector pSE180 (ATCC 209605) comprisingthe ermE gene of Sacc. erythraea inserted into the aveC ORF of S.avermitilis.

[0040]FIG. 4. BamHI restriction map of the avermectin polyketidesynthase gene cluster from S. avermitilis with five overlapping cosmidclones identified (i.e., pSE65, pSE66, pSE67, pSE68, pSE69). Therelationship of pSE118 and pSE119 is also indicated.

[0041]FIG. 5. HPLC analysis of fermentation products produced by S.avermitilis strains. Peak quantitation was performed by comparison tostandard quantities of cyclohexyl B1. Cyclohexyl B2 retention time was7.4-7.7 min; cyclohexyl B1 retention time was 11.9-12.3 min. FIG. 5A. S.avermmitilis strain SE180-11 with an inactivated aveC ORF. FIG. 5B. S.avermitilis strain SE180-11 transformed with pSE186 (ATCC 209604). FIG.5C. S. avermitilis strain SE180-11 transformed with pSE187. FIG. 5D. S.avermitilis strain SE180-11 transformed with pSE188.

[0042]FIG. 6. Comparison of deduced amino acid sequences encoded by theaveC ORF of S. avermitilis (SEQ ID NO:2), an aveC homolog partial ORFfrom S. griseochromogenes (SEQ ID NO:5), and the aveC homolog ORF fromS. hygroscopicus (SEQ ID NO:4). The valine residue in bold is theputative start site for the protein. Conserved residues are shown incapital letters for homology in all three sequences and in lower caseletters for homology in 2 of the 3 sequences. The amino acid sequencescontain approximately 50% sequence identity.

[0043]FIG. 7. Hybrid plasmid construct containing a 564 bp BsaAI/KpnIfragment from the S. hygroscopicus aveC homolog gene inserted into theBsaAI/KpnI site in the S. avermitilis aveC ORF.

5. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention relates to the identification andcharacterization of polynucleotide molecules having nucleotide sequencesthat encode the AveC gene product from Streptomyces avermitilis, theconstruction of novel strains of S. avermitilis that can be used toscreen mutated AveC gene products for their effect on avermectinproduction, and the discovery that certain mutated AveC gene productscan reduce the ratio of B2:B1 avermectins produced by S. avermitilis. Byway of example, the invention is described in the sections below for apolynucleotide molecule having either a nucleotide sequence that is thesame as the S. avermitilis AveC gene product-encoding sequence ofplasmid pSE186 (ATCC 209604), or the nucleotide sequence of the ORF ofFIG. 1 (SEQ ID NO:1), and for polynucleotides molecules having mutatednucleotide sequences derived therefrom and degenerate variants thereof.However, the principles set forth in the present invention can beanalogously applied to other polynucleotide molecules, including aveChomolog genes from other Streptomyces species including, e.g., S.hygroscopicus and S. griseochromogenes, among others.

5.1. Polynucleotide Molecules Encoding The S. avermitilis AveC GeneProduct

[0045] The present invention provides an isolated polynucleotidemolecule comprising the complete aveC ORF of S. avermitilis or asubstantial portion thereof, which isolated polynucleotide moleculelacks the next complete ORF that is located downstream from the aveC ORFin situ in the S. avermitilis chromosome.

[0046] The isolated polynucleotide molecule of the present inventionpreferably comprises a nucleotide sequence that is the same as the S.avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC209604), or that is the same as the nucleotide sequence of the ORF ofFIG. 1 (SEQ ID NO:1) or substantial portion thereof. As used herein, a“substantial portion” of an isolated polynucleotide molecule comprisinga nucleotide sequence encoding the S. avermitilis AveC gene productmeans an isolated polynucleotide molecule comprising at least about 70%of the complete aveC ORF sequence shown in FIG. 1 (SEQ ID NO:1), andthat encodes a functionally equivalent AveC gene product In this regard,a “functionally equivalent” AveC gene product is defined as a geneproduct that, when expressed in S. avermitilis strain ATCC 53692 inwhich the native aveC allele has been inactivated, results in theproduction of substantially the same ratio and amount of avermectins asproduced by S. avermitilis strain ATCC 53692 which instead expressesonly the wild-type, functional aveC allele native to S. avermitilisstrain ATCC 53692.

[0047] In addition to the nucleotide sequence of the aveC ORF, theisolated polynucleotide molecule of the present invention can furthercomprise nucleotide sequences that naturally flank the aveC gene in situin S. avermitilis, such as those flanking nucleotide sequences shown inFIG. 1 (SEQ ID NO:1).

[0048] The present invention further provides an isolated polynucleotidemolecule comprising the nucleotide sequence of SEQ ID NO:1 or adegenerate variant thereof.

[0049] As used herein, the terms “polynucleotide molecule,”“polynucleotide sequence,” “coding sequence,” “open-reading frame,” and“ORF” are intended to refer to both DNA and RNA molecules, which caneither be single-stranded or double-stranded, and that can betranscribed and translated (DNA), or translated (RNA), into an AveC geneproduct or, as described below, into an AveC homolog gene product, orinto a polypeptide that is homologous to an AveC gene product or AveChomolog gene product in an appropriate host cell expression system whenplaced under the control of appropriate regulatory elements. A codingsequence can include but is not limited to prokaryotic sequences, cDNAsequences, genomic DNA sequences, and chemically synthesized DNA and RNAsequences.

[0050] The nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) comprisesfour different GTG codons at bp positions 42, 174, 177 and 180. Asdescribed in Section 9 below, multiple deletions of the 540 region ofthe aveC ORF (FIG. 1; SEQ ID NO:1) were constructed to help define whichof these codons could function in the aveC ORF as start sites forprotein expression. Deletion of the first GTG site at bp 42 did noteliminate AveC activity. Additional deletion of all of the GTG codons atbp positions 174, 177 and 180 together eliminated AveC activity,indicating that this region is necessary for protein expression. Thepresent invention thus encompasses variable length aveC ORFs.

[0051] The present invention further provides a polynucleotide moleculehaving a nucleotide sequence that is homologous to the S. avermitilisAveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), orto the nucleotide sequence of the aveC ORF presented in FIG. 1 (SEQ IDNO:1) or substantial portion thereof. The term “homologous” when used torefer to a polynucleotide molecule that is homologous to an S.avermitilis AveC gene product-encoding sequence means a polynucleotidemolecule having a nucleotide sequence: (a) that encodes the same AveCgene product as the S. avermitilis AveC gene product-encoding sequenceof plasmid pSE186 (ATCC 209604), or that encodes the same AveC geneproduct as the nucleotide sequence of the aveC ORF presented in FIG. 1(SEQ ID NO:1), but that includes one or more silent changes to thenucleotide sequence according to the degeneracy of the genetic code(i.e., a degenerate variant); or (b) that hybridizes to the complementof a polynucleotide molecule having a nucleotide sequence that encodesthe amino acid sequence encoded by the AveC gene product-encodingsequence of plasmid pSE186 (ATCC 209604) or that encodes the amino acidsequence shown in FIG. 1 (SEQ ID NO:2) under moderately stringentconditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7%sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.2×SSC/0.1% SDS at 42° C. (see Ausubel et al. (eds.), 1989, CurrentProtocols in Molecular Biology, Vol. I, Green Publishing Associates,Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3), and encodesa functionally equivalent AveC gene product as defined above. In apreferred embodiment, the homologous polynucleotide molecule hybridizesto the complement of the AveC gene product-encoding nucleotide sequenceof plasmid pSE186 (ATCC 209604) or to the complement of the nucleotidesequence of the aveC ORF presented in FIG. 1 (SEQ ID NO:1) orsubstantial portion thereof under highly stringent conditions, i.e.,hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., 1989,above), and encodes a functionally equivalent AveC gene product asdefined above.

[0052] The activity of an AveC gene product and potential functionalequivalents thereof can be determined through HPLC analysis offermentation products, as described in the examples below.Polynucleotide molecules having nucleotide sequences that encodefunctional equivalents of the S. avermitilis AveC gene product includenaturally occurring aveC genes present in other strains of S.avermitilis, aveC homolog genes present in other species ofStreptomyces, and mutated aveC alleles, whether naturally occurring orengineered.

[0053] The present invention further provides a polynucleotide moleculecomprising a nucleotide sequence that encodes a polypeptide having anamino acid sequence that is homologous to the amino acid sequenceencoded by the AveC gene product-encoding sequence of plasmid pSE186(ATCC 209604), or the amino acid sequence of FIG. 1 (SEQ ID NO:2) orsubstantial portion thereof. As used herein, a “substantial portion” ofthe amino acid sequence of FIG. 1 (SEQ ID NO:2) means a polypeptidecomprising at least about 70% of the amino acid sequence shown in FIG. 1(SEQ ID NO:2), and that constitutes a functionally equivalent AveC geneproduct, as defined above.

[0054] As used herein to refer to amino acid sequences that arehomologous to the amino acid sequence of an AveC gene product from S.avermitilis, the term “homologous” refers to a polypeptide whichotherwise has the amino acid sequence of FIG. 1 (SEQ ID NO:2), but inwhich one or more amino acid residues has been conservativelysubstituted with a different amino acid residue, wherein said amino acidsequence has at least about 70%, more preferably at least about 80%, andmost preferably at least about 90% amino acid sequence identity to thepolypeptide encoded by the AveC gene product-encoding sequence ofplasmid pSE186 (ATCC 209604) or the amino acid sequence of FIG. 1 (SEQID NO:2) as determined by any standard amino acid sequence identityalgorithm, such as the BLASTP algorithm (GENBANK, NCBI), and where suchconservative substitution results in a functionally equivalent geneproduct, as defined above. Conservative amino acid substitutions arewell known in the art Rules for making such substitutions include thosedescribed by Dayhof, M. D., 1978, Nat. Biomed. Res. Found., Washington,D.C., Vol. 5, Sup. 3, among others. More specifically, conservativeamino acid substitutions are those that generally take place within afamily of amino acids that are related in the acidity or polarity.Genetically encoded amino acids are generally divided into four groups:(1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine;(3) non-polar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine.Phenylalanine, tryptophan and tyrosine are also jointly classified asaromatic amino acids. One or more replacements within any particulargroup, e.g., of a leucine with an isoleucine or valine, or of anaspartate with a glutamate, or of a threonine with a serine, or of anyother amino acid residue with a structurally related amino acid residue,e.g., an amino acid residue with similar acidity or polarity, or withsimilarity in some combination thereof, will generally have aninsignificant effect on the function of the polypeptide.

[0055] The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence encoding an AveC homolog geneproduct As used herein, an “AveC homolog gene product” is defined as agene product having at least about 50% amino acid sequence identity toan AveC gene product of S. avermitilis comprising the amino acidsequence encoded by the AveC gene product-encoding sequence of plasmidpSE186 (ATCC 209604), or the amino acid sequence shown in FIG. 1 (SEQ IDNO:2), as determined by any standard amino acid sequence identityalgorithm, such as the BLASTP algorithm (GENBANK, NCBI). In anon-limiting embodiment the AveC homolog gene product is from S.hygroscopicus, (described in EP application 0298423; deposit FERMBP-1901) and comprises the amino acid sequence of SEQ ID NO:4, or asubstantial portion thereof. A “substantial portion” of the amino acidsequence of SEQ ID NO:4 means a polypeptide comprising at least about70% of the amino acid sequence of SEQ ID NO:4, and that constitutes afunctionally equivalent AveC homolog gene product A “functionallyequivalent” AveC homolog gene product is defined as a gene product that,when expressed in S. hygroscopicus strain FERM BP-1901 in which thenative aveC homolog allele has been inactivated, results in theproduction of substantially the same ratio and amount of milbermycins asproduced by S. hygroscopicus strain FERM BP-1901 expressing instead onlythe wild-type, functional aveC homolog allele native to S. hygroscopicusstrain FERM BP-1901. In a non-limiting embodiment, the isolatedpolynucleotide molecule of the present invention that encodes the S.hygroscopicus AveC homolog gene product comprises the nucleotidesequence of SEQ ID NO:3 or a substantial portion thereof. In thisregard, a “substantial portion” of the isolated polynucleotide moleculecomprising the nucleotide sequence of SEQ ID NO:3 means an isolatedpolynucleotide molecule comprising at least about 70% of the nucleotidesequence of SEQ ID NO:3, and that encodes a functionally equivalent AveChomolog gene product, as defined immediately above.

[0056] The present invention further provides a polynucleotide moleculecomprising a nucleotide sequence that is homologous to the S.hygroscopicus nucleotide sequence of SEQ ID NO:3. The term “homologous”when used to refer to a polynucleotide molecule comprising a nucleotidesequence that is homologous to the S. hygroscopicus AveC homolog geneproduct-encoding sequence of SEQ ID NO:3 means a polynucleotide moleculehaving a nucleotide sequence: (a) that encodes the same gene product asthe nucleotide sequence of SEQ ID NO:3, but that includes one or moresilent changes to the nucleotide sequence according to the degeneracy ofthe genetic code (i.e., a degenerate variant); or (b) that hybridizes tothe complement of a polynucleotide molecule having a nucleotide sequencethat encodes the amino acid sequence of SEQ ID NO:4, under moderatelystringent conditions, i.e., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at42° C. (see Ausubel et al. above), and encodes a functionally equivalentAveC homolog gene product as defined above. In a preferred embodiment,the homologous polynucleotide molecule hybridizes to the complement ofthe AveC homolog gene product-encoding nucleotide sequence of SEQ IDNO:3, under highly stringent conditions, i.e., hybridization tofilter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al, 1989, above), andencodes a functionally equivalent AveC homolog gene product as definedabove.

[0057] The present invention further provides a polynucleotide moleculecomprising a nucleotide sequence that encodes a polypeptide that ishomologous to the S. hygroscopicus AveC homolog gene product. As usedherein to refer to polypeptides that are homologous to the AveC homologgene product of SEQ ID NO:4 from S. hygroscopicus, the term “homologous”refers to a polypeptide which otherwise has the amino acid sequence ofSEQ ID NO:4, but in which one or more amino acid residues has beenconservatively substituted with a different amino acid residue asdefined above, wherein said amino acid sequence has at least about 70%,more preferably at least about 80%, and most preferably at least about90% amino acid sequence identity to the polypeptide of SEQ ID NO:4, asdetermined by any standard amino acid sequence identity algorithm, suchas the BLASTP algorithm (GENBANK, NCBI), and where such conservativesubstitution results in a functionally equivalent AveC homolog geneproduct, as defined above.

[0058] The present invention further provides oligonucleotides thathybridize to a polynucleotide molecule having the nucleotide sequence ofFIG. 1 (SEQ ID NO:1) or SEQ ID NO:3, or to a polynucleotide moleculehaving a nucleotide sequence which is the complement of the nucleotidesequence of FIG. 1 (SEQ ID NO:1) or SEQ ID NO:3. Such oligonucleotidesare at least about 10 nucleotides in length, and preferably from about15 to about 30 nucleotides in length, and hybridize to one of theaforementioned polynucleotide molecules under highly stringentconditions, i.e., washing in 6×SSC/0.5% sodium pyrophosphate at ˜37° C.for ˜14-base oligos, at ˜48° C. for ˜17-base oligos, at ˜55° C. for˜20-base oligos, and at ˜60° C. for ˜23-base oligos. In a preferredembodiment, the oligonucleotides are complementary to a portion of oneof the aforementioned polynucleotide molecules. These oligonucleotidesare useful for a variety of purposes including to encode or act asantisense molecules useful in gene regulation, or as primers inamplification of aveC or aveC homolog-encoding polynucleotide molecules.

[0059] Additional aveC homolog genes can be identified in other speciesor strains of Streptomyces using the polynucleotide molecules oroligonucleotides disclosed herein in conjunction with known techniques.For example, an oligonucleotide molecule comprising a portion of the S.avermitilis nucleotide sequence of FIG. 1 (SEQ ID NO:1) or a portion ofthe S. hygroscopicus nucleotide sequence of SEQ ID NO:3 can bedetectably labeled and used to screen a genomic library constructed fromDNA derived from the organism of interest. The stringency of thehybridization conditions is selected based on the relationship of thereference organism, in this example S. avermitilis or S. hygroscopicus,to the organism of interest. Requirements for different stringencyconditions are well known to those of skill in the art, and suchconditions will vary predictably depending on the specific organismsfrom which the library and the labeled sequences are derived. Sucholigonucleotides are preferably at least about 15 nucleotides in lengthand include, e.g., those described in the examples below. Amplificationof homolog genes can be carried out using these and otheroligonucleotides by applying standard techniques such as the polymerasechain reaction (PCR), although other amplification techniques known inthe art, e.g., the ligase chain reaction, can also be used.

[0060] Clones identified as containing aveC homolog nucleotide sequencescan be tested for their ability to encode a functional AveC homolog geneproduct. For this purpose, the clones can be subjected to sequenceanalysis in order to identify a suitable reading frame, as well asinitiation and termination signals. Alternatively or additionally, thecloned DNA sequence can be inserted into an appropriate expressionvector, i.e., a vector that contains the necessary elements for thetranscription and translation of the inserted protein-coding sequence.Any of a variety of host/vector systems can be used as described below,including but not limited to bacterial systems such as plasmid,bacteriophage, or cosmid expression vectors. Appropriate host cellstransformed with such vectors comprising potential aveC homolog codingsequences can then be analyzed for AveC-type activity using methods suchas HPLC analysis of fermentation products, as described, e.g., inSection 7, below.

[0061] Production and manipulation of the polynucleotide moleculesdisclosed herein are within the skill in the art and can be carried outaccording to recombinant techniques described, e.g., in Maniatis, et.al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Ausubel, et al., 1989,Current Protocols In Molecular Biology, Greene Publishing Associates &Wiley Interscience, N.Y.; Sambrook, et al., 1989, Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Innis et al. (eds), 1995, PCR Strategies, AcademicPress, Inc., San Diego; and Erlich (ed), 1992, PCR Technology, OxfordUniversity Press, New York, all of which are incorporated herein byreference. Polynucleotide clones encoding AveC gene products or AveChomolog gene products can be identified using any method known in theart, including but not limited to the methods set forth in Section 7,below. Genomic DNA libraries can be screened for aveC and aveC homologcoding sequences using techniques such as the methods set forth inBenton and Davis, 1977, Science 196:180, for bacteriophage libraries,and in Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. USA,72:3961-3965, for plasmid libraries. Polynucleotide molecules havingnucleotide sequences known to include the aveC ORF, as present, e.g., inplasmid pSE186 (ATCC 209604), or in plasmid pSE119 (described in Section7, below), can be used as probes in these screening experiments.Alternatively, oligonucleotide probes can be synthesized that correspondto nucleotide sequences deduced from partial or complete amino acidsequences of the purified AveC homolog gene product

5.2. Recombinant Systems 5.2.1. Cloning And Expression Vectors

[0062] The present invention further provides recombinant cloningvectors and expression vectors which are useful in cloning or expressingpolynucleotide molecules of the present invention comprising, e.g., theaveC ORF of S. avermitilis or any aveC homolog ORFs. In a non-limitingembodiment, the present invention provides plasmid pSE186 (ATCC 209604),which comprises the complete ORF of the aveC gene of S. avermitilis.

[0063] All of the following description regarding the aveC ORF from S.avermitilis, or a polynucleotide molecule comprising the aveC ORF fromS. avermitilis or portion thereof, or an S. avermitilis AveC geneproduct, also refers to aveC homologs and AveC homolog gene products,unless indicated explicitly or by context.

[0064] A variety of different vectors have been developed for specificuse in Streptomyces, including phage, high copy number plasmids, lowcopy number plasmids, and E.coli-Streptomyces shuttle vectors, amongothers, and any of these can be used to practice the present invention.A number of drug resistance genes have also been cloned fromStreptomyces, and several of these genes have been incorporated intovectors as selectable markers. Examples of current vectors for use inStreptomyces are presented, among other places, in Hutchinson, 1980,Applied Biochem. Biotech. 16:169-190.

[0065] Recombinant vectors of the present invention, particularlyexpression vectors, are preferably constructed so that the codingsequence for the polynucleotide molecule of the invention is inoperative association with one or more regulatory elements necessary fortranscription and translation of the coding sequence to produce apolypeptide. As used herein, the term “regulatory element” includes butis not limited to nucleotide sequences that encode inducible andnon-inducible promoters, enhancers, operators and other elements knownin the art that serve to drive and/or regulate expression ofpolynucleotide coding sequences. Also, as used herein, the codingsequence is in “operative association” with one or more regulatoryelements where the regulatory elements effectively regulate and allowfor the transcription of the coding sequence or the translation of itsmRNA, or both.

[0066] Typical plasmid vectors that can be engineered to contain apolynucleotide molecule of the present invention include pCR-Blunt,pCR2.1 (Invitrogen), pGEM3Zf (Promega), and the shuttle vector pWHM3(Vara et al., 1989, J. Bact. 171:5872-5881), among many others.

[0067] Methods are well-known in the art for constructing recombinantvectors containing particular coding sequences in operative associationwith appropriate regulatory elements, and these can be used to practicethe present invention. These methods include in vitro recombinanttechniques, synthetic techniques, and in vivo genetic recombination.See, e.g., the techniques described in Maniatis et al., 1989, above;Ausubel et al, 1989, above; Sambrook et al., 1989, above; Innis et al.,1995, above; and Erlich, 1992, above.

[0068] The regulatory elements of these vectors can vary in theirstrength and specificities. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationelements can be used. Non-limiting examples of transcriptionalregulatory regions or promoters for bacteria include the β-gal promoter,the T7 promoter, the TAC promoter, λ left and right promoters, trp andlac promoters, trp-lac fusion promoters and, more specifically forStreptomyces, the promoters ermE, melC, and tipA, etc. In a specificembodiment described in Section 11 below, an expression vector wasgenerated that contained the aveC ORF cloned adjacent to the strongconstitutive ermE promoter from Saccharopolyspora erythraea. The vectorwas transformed into S. avermitilis, and subsequent HPLC analysis offermentation products indicated an increased titer of avermectinsproduced compared to production by the same strain but which insteadexpresses the wild-type aveC allele.

[0069] Fusion protein expression vectors can be used to express an AveCgene product-fusion protein. The purified fusion protein can be used toraise antisera against the AveC gene product, to study the biochemicalproperties of the AveC gene product, to engineer AveC fusion proteinswith different biochemical activities, or to aid in the identificationor purification of the expressed AveC gene product. Possible fusionprotein expression vectors include but are not limited to vectorsincorporating sequences that encode β-galactosidase and trpE fusions,maltose-binding protein fusions, glutathione-S-transferase fusions andpolyhistidine fusions (carrier regions). In an alternative embodiment,an AveC gene product or a portion thereof can be fused to an AveChomolog gene product, or portion thereof, derived from another speciesor strain of Streptomyces, such as, e.g., S. hygroscopicus or S.griseochromogenes. In a particular embodiment described in Section 12,below, and depicted in FIG. 7, a chimeric plasmid was constructed thatcontains a 564 bp region of the S. hygroscopicus aveC homolog ORFreplacing a homologous 564 bp region of the S. avermitilis aveC ORF.Such hybrid vectors can be transformed into S. avermitilis cells andtested to determine their effect, e.g., on the ratio of class 2:1avermectin produced.

[0070] AveC fusion proteins can be engineered to comprise a regionuseful for purification. For example, AveC-maltose-binding proteinfusions can be purified using amylose resin;AveC-glutathione-S-transferase fusion proteins can be purified usingglutathione-agarose beads; and AveC-polyhistidine fusions can be,purified using divalent nickel resin. Alternatively, antibodies againsta carrier protein or peptide can be used for affinity chromatographypurification of the fusion protein. For example, a nucleotide sequencecoding for the target epitope of a monoclonal antibody can be engineeredinto the expression vector inoperative association with the regulatoryelements and situated so that the expressed epitope is fused to the AveCpolypeptide. For example, a nucleotide sequence coding for the FLAG™epitope tag (International Biotechnologies Inc.), which is a hydrophilicmarker peptide, can be inserted by standard techniques into theexpression vector at a point corresponding, e.g., to the carboxylterminus of the AveC polypeptide. The expressed AveC polypeptide-FLAG™epitope fusion product can then be detected and affinity-purified usingcommercially available anti-FLAG antibodies.

[0071] The expression vector encoding the AveC fusion protein can alsobe engineered to contain polylinker sequences that encode specificprotease cleavage sites so that the expressed AveC polypeptide can bereleased from the carrier region or fusion partner by treatment with aspecific protease. For example, the fusion protein vector can includeDNA sequences encoding thrombin or factor Xa cleavage sites, amongothers.

[0072] A signal sequence upstream from, and in reading frame with, theaveC ORF can be engineered into the expression vector by known methodsto direct the trafficking and secretion of the expressed gene product.Non-limiting examples of signal sequences include those from α-factor,immunoglobulins, outer membrane proteins, penicillinase, and T-cellreceptors, among others.

[0073] To aid in the selection of host cells transformed or transfectedwith cloning or expression vectors of the present invention, the vectorcan be engineered to further comprise a coding sequence for a reportergene product or other selectable marker. Such a coding sequence ispreferably in operative association with the regulatory element codingsequences, as described above. Reporter genes that are useful in theinvention are well- known in the art and include those encoding greenfluorescent protein, luciferase, xylE, and tyrosinase, among others.Nucleotide sequences encoding selectable markers are well known in theart, and include those that encode gene products conferring resistanceto antibiotics or anti-metabolites, or that supply an auxotrophicrequirement Examples of such sequences include those that encoderesistance to erythromycin, thiostrepton or kanamycin, among manyothers.

5.2.2. Transformation Of Host Cells

[0074] The present invention further provides transformed host cellscomprising a polynucleotide molecule or recombinant vector of theinvention, and novel strains or cell lines derived therefrom. Host cellsuseful in the practice of the invention are preferably Streptomycescells, although other prokaryotic cells or eukaryotic cells can also beused. Such transformed host cells typically include but are not limitedto microorganisms, such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA vectors, or yeasttransformed with recombinant vectors, among others.

[0075] The polynucleotide molecules of the present invention areintended to function in Streptomyces cells, but can also be transformedinto other bacterial or eukaryotic cells, e.g., for cloning orexpression purposes. A strain of E. coli can typically be used, such as,e.g., the DH5α strain, available from the American Type CultureCollection (ATCC), Rockville, Md., USA (Accession No. 31343), and fromcommercial sources (Stratagene). Preferred eukaryotic host cells includeyeast cells, although mammalian cells or insect cells can also beutilized effectively.

[0076] The recombinant expression vector of the invention is preferablytransformed or transfected into one or more host cells of asubstantially homogeneous culture of cells. The expression vector isgenerally introduced into host cells in accordance with knowntechniques, such as, e.g., by protoplast transformation, calciumphosphate precipitation, calcium chloride treatment, microinjection,electroporation, transfection by contact with a recombined virus,liposome-mediated transfection, DEAE-dextran transfection, transduction,conjugation, or microprojectile bombardment. Selection of transformantscan be conducted by standard procedures, such as by selecting for cellsexpressing a selectable marker, e.g., antibiotic resistance, associatedwith the recombinant vector, as described above.

[0077] Once the expression vector is introduced into the host cell, theintegration and maintenance of the avec coding sequence either in thehost cell chromosome or episomally can be confirmed by standardtechniques, e.g., by Southern hybridization analysis, restriction enzymeanalysis, PCR analysis, including reverse transcriptase PCR (rt-PCR), orby immunological assay to detect the expected gene product Host cellscontaining and/or expressing the recombinant aveC coding sequence can beidentified by any of at least four general approaches which arewell-known in the art, including: (i) DNA-DNA, DNA-RNA, or RNA-antisenseRNA hybridization; (ii) detecting the presence of “marker” genefunctions; (iii) assessing the level of transcription as measured by theexpression of aveC-specific mRNA transcripts in the host cell; and (iv)detecting the presence of mature polypeptide product as measured, e.g.,by immunoassay or by the presence of AveC biological activity (e.g., theproduction of specific ratios and amounts of avermectins indicative ofAveC activity in, e.g., S. avermitilis host cells).

5.2.3. Expression And Characterization Of A Recombinant AveC GeneProduct

[0078] Once the aveC coding sequence has been stably introduced into anappropriate host cell, the transformed host cell is clonally propagated,and the resulting cells can be grown under conditions conducive to themaximum production of the AveC gene product Such conditions typicallyinclude growing cells to high density. Where the expression vectorcomprises an inducible promoter, appropriate induction conditions suchas, e.g., temperature shift, exhaustion of nutrients, addition ofgratuitous inducers (e.g., analogs of carbohydrates, such asisopropyl-β-D-thiogalactopyranoside (IPTG)), accumulation of excessmetabolic by-products, or the like, are employed as needed to induceexpression.

[0079] Where the expressed AveC gene product is retained inside the hostcells, the cells are harvested and lysed, and the product isolated andpurified from the lysate under extraction conditions known in the art tominimize protein degradation such as, e.g., at 4° C., or in the presenceof protease inhibitors, or both. Where the expressed AveC gene productis secreted from the host cells, the exhausted nutrient medium cansimply be collected and the product isolated therefrom.

[0080] The expressed AveC gene product can be isolated or substantiallypurified from cell lysates or culture medium, as appropriate, usingstandard methods, including but not limited to any combination of thefollowing methods: ammonium sulfate precipitation, size fractionation,ion exchange chromatography, HPLC, density centrifugation, and affinitychromatography. Where the expressed AveC gene product exhibitsbiological activity, increasing purity of the preparation can bemonitored at each step of the purification procedure by use of anappropriate assay. Whether or not the expressed AveC gene productexhibits biological activity, it can be detected as based, e.g., onsize, or reactivity with an antibody otherwise specific for AveC, or bythe presence of a fusion tag. As used herein, an AveC gene product is“substantially purified” where the product constitutes more than about20 wt % of the protein in a particular preparation. Also, as usedherein, an AveC gene product is “isolated” where the product constitutesat least about 80 wt % of the protein in a particular preparation.

[0081] The present invention thus provides a recombinantly-expressedisolated or substantially purified S. avermitilis AveC gene productcomprising the amino acid sequence encoded by the AveC geneproduct-encoding sequence of plasmid pSE186 (ATCC 209604), or the aminoacid sequence of FIG. 1 (SEQ ID NO:2) or a substantial portion thereof,and homologs thereof.

[0082] The present invention further provides a recombinantly-expressedisolated or substantially purified S. hygroscopicus AveC homolog geneproduct comprising the amino acid sequence of SEQ ID NO:4 or asubstantial portion thereof, and homologs thereof.

[0083] The present invention further provides a method for producing anAveC gene product, comprising culturing a host cell transformed with arecombinant expression vector, said vector comprising a polynucleotidemolecule having a nucleotide sequence encoding the AveC gene product,which polynucleotide molecule is in operative association with one ormore regulatory elements that control expression of the polynucleotidemolecule in the host cell, under conditions conducive to the productionof the recombinant AveC gene product, and recovering the AveC geneproduct from the cell culture.

[0084] The recombinantiy expressed S. avermitilis AveC gene product isuseful for a variety of purposes, including for screening compounds thatalter AveC gene product function and thereby modulate avermectinbiosynthesis, and for raising antibodies directed against the AveC geneproduct.

[0085] Once an AveC gene product of sufficient purity has been obtained,it can be characterized by standard methods, including by SDS-PAGE, sizeexclusion chromatography, amino acid sequence analysis, biologicalactivity in producing appropriate products in the avermectinbiosynthetic pathway, etc. For example, the amino acid sequence of theAveC gene product can be determined using standard peptide sequencingtechniques. The AveC gene product can be further characterized usinghydrophilicity analysis (see, e.g., Hopp and Woods, 1981, Proc. Natl.Acad. Sci. USA 78:3824), or analogous software algorithms, to identifyhydrophobic and hydrophilic regions of the AveC gene product. Structuralanalysis can be carried out to identify regions of the AveC gene productthat assume specific secondary structures. Biophysical methods such asX-ray crystallography (Engstrom, 1974, Biochem. Exp. Biol. 11: 7-13),computer modelling (Fletterick and Zoller (eds), 1986, in: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.), and nuclear magnetic resonance (NMR) can be usedto map and study sites of interaction between the AveC gene product andits substrate. Information obtained from these studies can be used toselect new sites for mutation in the aveC ORF to help develop newstrains of S. avermitilis having more desirable avermectin productioncharacteristics.

5.3. Construction And Use Of AveC Mutants

[0086] The present invention provides a polynucleotide moleculecomprising a nucleotide sequence that is otherwise the same as the S.avermitilis aveC allele or a degenerate variant thereof, or the AveCgene product-encoding sequence of plasmid pSE1 86 (ATCC 209604) or adegenerate variant thereof, or the nucleotide sequence of the aveC ORFof S. avermitilis as presented in FIG. 1 (SEQ ID NO:1) or a degeneratevariant thereof, but that further comprises one or more mutations, sothat cells of S. avermitilis strain ATCC 53692 in which the wild-typeaveC allele has been inactivated and that express the polynucleotidemolecule comprising the mutated nucleotide sequence or the degeneratevariant thereof produce a different ratio or amount of avermectins thanare produced by cells of S. avermitilis strain ATCC 53692 that insteadexpress only the wild-type aveC allele.

[0087] According to the present invention, such polynucleotide moleculescan be used to produce novel strains of S. avermitilis that exhibit adetectable change in avermectin production compared to the same strainwhich instead expresses only the wild-type aveC allele. In a preferredembodiment, such polynucleotide molecules are useful to produce novelstrains of S. avermitilis that produce avermectins in a reduced class2:1 ratio compared to the same strain which instead expresses only thewild-type aveC allele. In a further preferred embodiment, suchpolynucleotide molecules are useful to produce novel strains of S.avermitilis that produce increased levels of avermectins compared to thesame strain which instead expresses only a single wild-type aveC allele.In a further preferred embodiment, such polynucleotide molecules areuseful to produce novel strains of S. avermitilis in which the aveC genehas been inactivated.

[0088] Mutations to the aveC allele or coding sequence include anymutations that introduce one or more amino acid deletions, additions, orsubstitutions into the AveC gene product, or that result in truncationof the AveC gene product, or any combination thereof, and that producethe desired result. Such mutated avec allele sequences are also intendedto include any degenerate variants thereof. For example, the presentinvention provides polynucleotide molecules comprising the nucleotidesequence of the aveC allele or a degenerate variant thereof, or the AveCgene product-encoding sequence of plasmid pSE186 (ATCC 209604) or adegenerate variant thereof, or the nucleotide sequence of the aveC ORFof S. avermitilis as present in FIG. 1 (SEQ ID NO:1) or a degeneratevariant thereof, but that further comprise one or more mutations thatencode the substitution of an amino acid residue with a different aminoacid residue at selected positions in the AveC gene product In severalnon-limiting embodiments, several of which are exemplified below, suchsubstitutions can be carried out at any amino acid positions of the AveCgene product which correspond to amino acid positions 38, 48, 55, 89,99, 111, 136, 138, 139, 154, 179, 228, 230, 238, 266, 275, 289 or 298 ofSEQ ID NO:2, or some combination thereof.

[0089] Mutations to the aveC coding sequence are carried out by any of avariety of known methods, including by use of error-prone PCR, or bycassette mutagenesis. For example, oligonucleotide-directed mutagenesiscan be employed to alter the sequence of the aveC allele or ORF in adefined way such as, e.g., to introduce one or more restriction sites,or a termination codon, into specific regions within the aveC allele orORF. Methods such as those described in U.S. Pat. No. 5,605,793, U.S.Pat. No. 5,830,721 and U.S. Pat. No. 5,837,458, which involve randomfragmentation, repeated cycles of mutagenesis, and nucleotide shuffling,can also be used to generate large libraries of polynucleotides havingnucleotide sequences encoding aveC mutations.

[0090] Targeted mutations can be useful, particularly where they serveto alter one or more conserved amino acid residues in the AveC geneproduct For example, a comparison of deduced amino acid sequences ofAveC gene products and AveC homolog gene products from S. avermitilis(SEQ ID NO:2), S. griseochromogenes (SEQ ID NO:5), and S. hygroscopicus(SEQ ID NO:4), as presented in FIG. 6, indicates sites of significantconservation of amino acid residues between these species. Targetedmutagenesis that leads to a change in one or more of these conservedamino acid residues can be particularly effective in producing novelmutant strains that exhibit desirable alterations in avermectinproduction.

[0091] Random mutagenesis can also be useful, and can be carried out byexposing cells of S. avermitilis to ultraviolet radiation or x-rays, orto chemical mutagens such as N-methyl-N′-nitrosoguanidine, ethyl methanesulfonate, nitrous acid or nitrogen mustards. See, e.g., Ausubel, 1989,above, for a review of mutagenesis techniques.

[0092] Once mutated polynucleotide molecules are generated, they arescreened to determine whether they can modulate avermectin biosynthesisin S. avermitilis. In a preferred embodiment, a polynucleotide moleculehaving a mutated nucleotide sequence is tested by complementing a strainof S. avermitilis in which the aveC gene has been inactivated to give anaveC negative (aveC) background. In a non-limiting method, the mutatedpolynucleotide molecule is spliced into an expression plasmid inoperative association with one or more regulatory elements, whichplasmid also preferably comprises one or more drug resistance genes toallow for selection of transformed cells. This vector is thentransformed into aveC host cells using known techniques, and transformedcells are selected and cultured in appropriate fermentation media underconditions that permit or induce avermectin production. Fermentationproducts are then analyzed by HPLC to determine the ability of themutated polynucleotide molecule to complement the host cell. Severalvectors bearing mutated polynucleotide molecules capable of reducing theB2:B1 ratio of avermectins, including pSE188, pSE199, pSE231, pSE239,and pSE290 through pSE297, are exemplified in Section 8:3, below.

[0093] The present invention provides methods for identifying mutationsof the S. avermitilis aveC ORF capable of altering the ratio and/oramount of avermectins produced. In a preferred embodiment, the presentinvention provides a method for identifying mutations of the aveC ORFcapable of altering the class 2:1 ratio of avermectins produced,comprising:

[0094] (a) determining the class 2:1 ratio of avermectins produced bycells of a strain of S. avermitilis in which the aveC allele nativethereto has been inactivated, and into which a polynucleotide moleculecomprising a nucleotide sequence encoding a mutated AveC gene producthas been introduced and is being expressed;

[0095] (b) determining the class 2:1 ratio of avermectins produced bycells of the same strain of S. avermitilis as in step (a) but whichinstead express only a wild-type aveC allele or an aveC allele havingthe nucleotide sequence of the ORF of FIG. 1 (SEQ ID NO:1) or anucleotide sequence that is homologous thereto; and

[0096] (c) comparing the class 2:1 ratio of avermectins produced by theS. avermitilis cells of step (a) to the class 2:1 ratio of avermectinsproduced by the S. avermitilis cells of step (b); such that if the class2:1 ratio of avermectins produced by the S. avermitilis cells of step(a) is different from the class 2:1 ratio of avermectins produced by theS. avermitilis cells of step (b), then a mutation of the aveC ORFcapable of altering the class 2:1 ratio of avermectins has beenidentified. In a preferred embodiment, the class 2:1 ratio ofavermectins is reduced by the mutation.

[0097] In a further preferred embodiment, the present invention providesa method for identifying mutations of the aveC ORF or genetic constructscomprising the aveC ORF capable of altering the amount of avermectinsproduced, comprising:

[0098] (a) determining the amount of avermectins produced by cells of astrain of S. avermitilis in which the aveC allele native thereto hasbeen inactivated, and into which a polynucleotide molecule comprising anucleotide sequence encoding a mutated AveC gene product or comprising agenetic construct comprising a nucleotide sequence encoding an AveC geneproduct has been introduced and is being expressed;

[0099] (b) determining the amount of avermectins produced by cells ofthe same strain of S. avermitilis as in step (a) but which insteadexpress only a wild-type aveC allele or a nucleotide sequence that ishomologous thereto; and

[0100] (c) comparing the amount of avermectins produced by the S.avermitilis cells of step (a) to the amount of avermectins produced bythe S. avermitilis cells of step (b); such that if the amount ofavermectins produced by the S. avermitilis cells of step (a) isdifferent from the amount of avermectins produced by the S. avermitiliscells of step (b), then a mutation of the aveC ORF or a geneticconstruct capable of altering the amount of avermectins has beenidentified. In a preferred embodiment, the amount of avermectinsproduced is increased by the mutation.

[0101] Any of the aforementioned methods for identifying mutations arebe carried out using fermentation culture media preferably supplementedwith cyclohexane carboxylic acid, although other appropriate fatty acidprecursors, such as any one of the fatty acid precursors listed in TABLE1, can also used.

[0102] Once a mutated polynucleotide molecule that modulates avermectinproduction in a desirable direction has been identified, the location ofthe mutation in the nucleotide sequence can be determined. For example,a polynucleotide molecule having a nucleotide sequence encoding amutated AveC gene product can be isolated by PCR and subjected to DNAsequence analysis using known methods. By comparing the DNA sequence ofthe mutated aveC allele to that of the wild-type aveC allele, themutation(s) responsible for the alteration in avermectin production canbe determined. In specific though non-limiting embodiments of thepresent invention, S. avermitilis AveC gene products comprising eithersingle amino acid substitutions at any of residues 55 (S55F), 138(S138T), 139 (A139T), or 230 (G230D), or double substitutions atpositions 138 (S138T) and 139 (A139T or A139F), yielded changes in AveCgene product function such that the ratio of class 2:1 avermectinsproduced was altered (see Section 8, below), wherein the recited aminoacid positions correspond to those presented in FIG. 1 (SEQ ID NO:2). Inaddition, the following seven combinations of mutations have each beenshown to effectively reduce the class 2:1 ratio of avermectins: (1)D48E/A89T; (2) S138T/A139T/G179S; (3) Q38P/L136P/E238D; (4)F99S/S138T/A139T/G179S; (5) A139T/M228T; (6) G111V/P289L; (7)A139T/K154E/Q298H. As used herein, the aforementioned designations, suchas A139T, indicate the original amino acid residue by single letterdesignation, which in this example is alanine (A), at the indicatedposition, which in this example is position 139 (referring to SEQ IDNO:2) of the polypeptide, followed by the amino acid residue whichreplaces the original amino acid residue, which in this example isthreonine (T). Accordingly, polynucleotide molecules having nucleotidesequences that encode mutated S. avermitilis AveC gene productscomprising amino acid substitutions or deletions at one or more of aminoacid positions 38, 48, 55, 89, 99, 111, 136, 138, 139, 154, 179, 228,230, 238, 266, 275, 289 or 298 (see FIG. 1), or any combination thereof,are encompassed by the present invention.

[0103] In a preferred embodiment, such mutations encode amino acidsubstitutions selected from one or more of the group consisting of:

[0104] (a) amino acid residue Q at position 38 replaced by P or by anamino acid that is a conservative substitution for P;

[0105] (b) amino acid residue D at position 48 replaced by E or by anamino acid that is a conservative substitution for E;

[0106] (c) amino acid residue A at position 89 replaced by T or by anamino acid that is a conservative substitution for T;

[0107] (d) amino acid residue F at position 99 replaced by S or by anamino acid that is a conservative substitution for S;

[0108] (e) amino acid residue G at position 111 replaced by V or by anamino acid that is a conservative substitution for V;

[0109] (f) amino acid residue L at position 136 replaced by P or by anamino acid that is a conservative substitution for P;

[0110] (g) amino acid residue S at position 138 replaced by T or by anamino acid that is a conservative substitution for T;

[0111] (h) amino acid residue A at position 139 replaced by T or F, orby an amino acid that is a conservative substitution for T or F;

[0112] (i) amino acid residue K at position 154 replaced by E or by anamino acid that is a conservative substitution for E;

[0113] (j) amino acid residue G at position 179 replaced by S or by anamino acid that is a conservative substitution for S;

[0114] (k) amino acid residue M at position 228 replaced by T or by anamino acid that is a conservative substitution for T;

[0115] (l) amino acid residue E at position 238 replaced by D or by anamino acid that is a conservative substitution for D;

[0116] (m) amino acid residue P at position 289 replaced by L or by anamino acid that is a conservative substitution for L; and

[0117] (n) amino acid residue Q at position 298 replaced by H or by anamino acid that is a conservative substitution for H;

[0118] wherein conservative amino acid substitutions are as definedabove in Section 5.1.

[0119] In a further preferred embodiment, such mutations encode acombination of amino acid substitutions, wherein the combination ofamino acid residues substituted is selected from the group consistingof:

[0120] (a) amino acid residues S138 and A139;

[0121] (b) amino acid residues D48 and A89;

[0122] (c) amino acid residues S138, A139 and G179;

[0123] (d) amino acid residues Q38, L136 and E238;

[0124] (e) amino acid residues F99, S138, A139 and G179;

[0125] (f) amino acid residues A139 and M228;

[0126] (g) amino acid residues G111 and P289; and

[0127] (h) amino acid residues A139, K154 and Q298.

[0128] In a further preferred embodiment, specific combinations ofmutations in the aveC allele useful in effectively reducing the class2:1 ratio of avermectins according to the present invention are selectedfrom one or more of the group consisting of:

[0129] (a) S138T/A139T

[0130] (b) S138T/A139F

[0131] (c) D48E/A89T;

[0132] (d) S138T/A139T/G179S;

[0133] (e) Q38P/L136P/E238D;

[0134] (f) F99S/S138T/A139T/G179S;

[0135] (g) A139T/M228T;

[0136] (h) G111V/P289L; and

[0137] (i) A139T/K154E/Q298H.

[0138] The present invention further provides compositions for makingnovel strains of S. avermitilis, the cells of which contain a mutatedaveC allele that results in the alteration of avermectin production. Forexample, the present invention provides recombinant vectors that can beused to target any of the polynucleotide molecules comprising mutatednucleotide sequences of the present invention to the site of the aveCgene of the S. avermitilis chromosome to either insert into or replacethe aveC ORF or a portion thereof by homologous recombination. Accordingto the present invention, however, a polynucleotide molecule comprisinga mutated nucleotide sequence of the present invention provided herewithcan also function to modulate avermectin biosynthesis when inserted intothe S. avermitilis chromosome at a site other than at the aveC gene, orwhen maintained episomally in S. avermitilis cells. Thus, the presentinvention also provides vectors comprising a polynucleotide moleculecomprising a mutated nucleotide sequence of the present invention, whichvectors can be used to insert the polynucleotide molecule at a site inthe S. avermitilis chromosome other than at the aveC gene, or to bemaintained episomally.

[0139] In a preferred embodiment, the present invention provides genereplacement vectors that can be used to insert a mutated aveC allele ordegenerate variant thereof into cells of a strain of S. avermitilis,thereby generating novel strains of S. avermitilis, the cells of whichproduce avermectins in an altered class 2:1 ratio compared to cells ofthe same strain which instead express only the wild-type aveC allele. Ina preferred embodiment, the class 2:1 ratio of avermectins produced bythe cells is reduced. Such gene replacement vectors can be constructedusing mutated polynucleotide molecules present in expression vectorsprovided herewith, such as, e.g., pSE188, pSE199, and pSE231, whichexpression vectors are exemplified in Section 8 below.

[0140] In a further preferred embodiment, the present invention providesvectors that can be used to insert a mutated aveC allele or degeneratevariant thereof into cells of a strain of S. avermitilis to generatenovel strains of cells that produce altered amounts of avermectinscompared to cells of the same strain which instead express only thewild-type aveC allele. In a preferred embodiment, the amount ofavermectins produced by the cells is increased. In a specific thoughnon-limiting embodiment, such a vector further comprises a strongpromoter as known in the art, such as, e.g., the strong constitutiveermE promoter from Saccharopolyspora erythraea, that is situatedupstream from, and in operative association with, the aveC allele. Sucha vector can be plasmid pSE189, described in Example 11 below, or can beconstructed using the mutated aveC allele of plasmid pSE189.

[0141] In a further preferred embodiment, the present invention providesgene replacement vectors that are useful to inactivate the aveC gene ina wild-type strain of S. avermitilis. In a non-limiting embodiment, suchgene replacement vectors can be constructed using the mutatedpolynucleotide molecule present in plasmid pSE180 (ATCC 209605), whichis exemplified in Section 8.1, below (FIG. 3). The present inventionfurther provides gene replacement vectors that comprise a polynucleotidemolecule comprising or consisting of nucleotide sequences that naturallyflank the aveC gene in situ in the S. avermitilis chromosome, including,e.g., those flanking nucleotide sequences shown in FIG. 1 (SEQ ID NO:1),which vectors can be used to delete the S. avermitilis aveC ORF.

[0142] The present invention further provides methods for making novelstrains of S. avermitilis comprising cells that express a mutated aveCallele and that produce an altered ratio and/or amount of avermectinscompared to cells of the same strain of S. avermitilis that insteadexpress only the wild-type aveC allele. In a preferred embodiment, thepresent invention provides a method for making novel strains of S.avermitilis comprising cells that express a mutated aveC allele and thatproduce an altered class 2:1 ratio of avermectins compared to cells ofthe same strain of S. avermitilis that instead express only a wild-typeaveC allele, comprising transforming cells of a strain of S. avermitiliswith a vector that carries a mutated aveC allele that encodes a geneproduct that alters the class 2:1 ratio of avermectins produced by cellsof a strain of S. avermitilis expressing the mutated aveC allele thereofcompared to cells of the same strain that instead express only awild-type aveC allele, and selecting transformed cells that produceavermectins in an altered class 2:1 ratio compared to the class 2:1ratio produced by cells of the strain that instead express only thewild-type aveC allele. In a more preferred embodiment, the presentinvention provides a method for making a novel strain of S. avermitilis,comprising transforming cells of a strain of S. avermitilis with avector capable of introducing a mutation into the aveC allele of suchcells, wherein the mutation to the aveC allele results in thesubstitution in the encoded AveC gene product of a different amino acidresidue at one or more amino acid positions corresponding to amino acidresidues 38, 48, 55, 89, 99, 111, 136, 138, 139, 154, 179, 228, 230,238, 266, 275, 289 or 298 of SEQ ID NO:2, such that cells of the S.avermitilis strain in which the aveC allele has been so mutated producea class 2:1 ratio of avermectins that is different from the ratioproduced by cells of the same S. avermitilis strain that instead expressonly the wild-type aveC allele. In a preferred embodiment, the alteredclass2:1 ratio of avermectins is reduced.

[0143] As used herein, where an amino acid residue encoded by an aveCallele in the S. avermitilis chromosome, or in a vector or isolatedpolynucleotide molecule of the present invention is referred to as“corresponding to” a particular amino acid residue of SEQ ID NO:2, orwhere an amino acid substitution is referred to as occurring at aparticular position “corresponding to” that of a specific numbered aminoacid residue of SEQ ID NO:2, this is intended to refer to the amino acidresidue at the same relative location in the AveC gene product, whichthe skilled artisan can quickly determine by reference to the amino acidsequence presented herein as SEQ ID NO:2.

[0144] The present invention further provides methods of making novelstrains wherein specific mutations in the aveC allele encodingparticular mutations are recited as base changes at specific nucleotidepositions in the avec allele “corresponding to” particular nucleotidepositions as shown in SEQ ID NO:1. As above with regard to correspondingamino acid positions, where a nucleotide position in the aveC allele isreferred to as “corresponding to” a particular nucleotide position inSEQ ID NO:1, this is intended to refer to the nucleotide at the samerelative location in the aveC nucleotide sequence, which the skilledartisan can quickly determine by reference to the nucleotide sequencepresented herein as SEQ ID NO:1.

[0145] In a further preferred embodiment, the present invention providesa method for making novel strains of S. avermitilis comprising cellsthat produce altered amounts of avermectin, comprising transformingcells of a strain of S. avermitilis with a vector that carries a mutatedaveC allele or a genetic construct comprising the aveC allele, theexpression of which results in an alteration in the amount ofavermectins produced by cells of a strain of S. avermitilis expressingthe mutated aveC allele or genetic construct as compared to cells of thesame strain that instead express only a single wild-type aveC allele,and selecting transformed cells that produce avermectins in an alteredamount compared to the amount of avermectins produced by cells of thestrain that instead express only the single wild-type aveC allele. In apreferred embodiment, the amount of avermectins produced in thetransformed cells is increased.

[0146] In a further preferred embodiment, the present invention providesa method for making novel strains of S. avermitilis, the cells of whichcomprise an inactivated aveC allele, comprising transforming cells of astrain of S. avermitilis that express any aveC allele with a vector thatinactivates the aveC allele, and selecting transformed cells in whichthe aveC allele has been inactivated. In a preferred though non-limitingembodiment, cells of a strain of S. avermitilis are transformed with agene replacement vector that carries an aveC allele that has beeninactivated by mutation or by replacement of a portion of the aveCallele with a heterologous gene sequence, and transformed cells areselected in which the aveC allele otherwise native thereto has beenreplaced with the inactivated aveC allele. Inactivation of the aveCallele can be determined by HPLC analysis of fermentation products, asdescribed below. In a specific though non-limiting embodiment describedin Section 8.1 below, the aveC allele is inactivated by insertion of theermE gene from Saccharopolyspora erythraea into the aveC ORF.

[0147] The present invention further provides novel strains of S.avermitilis comprising cells that have been transformed with any of thepolynucleotide molecules or vectors of the present invention. In apreferred embodiment, the present invention provides novel strains of S.avermitilis comprising cells which express a mutated aveC allele ordegenerate variant thereof in place of, or in addition to, the wild-typeaveC allele, wherein the cells of the novel strain produce avermectinsin an altered class 2:1 ratio compared to the class 2:1 ratio ofavermectins produced by cells of the same strain that instead expressonly the wild-type aveC allele. In a preferred embodiment, the alteredclass 2:1 ratio produced by the novel cells is reduced. Such novelstrains are useful in the large-scale production of commerciallydesirable avermectins such as doramectin. In a more preferredembodiment, the present invention provides cells of S. avermitiliscomprising any of the aforementioned mutations or combinations ofmutations in the aveC allele at nucleotide positions corresponding tothose presented hereinabove or which otherwise encode any of theaforementioned amino acid substitutions in the AveC gene productAlthough such mutations can be present in such cells on anextrachromosomal element such as a plasmid, it is preferred that suchmutations are present in the aveC allele located on the S. avermitilischromosome. In a preferred embodiment, the present invention provides astrain of Streptomyces avermitilis comprising cells having a mutation inthe aveC allele that encodes an AveC gene product having a substitutionat one or more amino acid positions corresponding to amino acid residues38, 48, 55, 89, 99, 111, 136, 138, 139, 154, 179, 228, 230, 238, 266,275, 289, or 298 of SEQ ID NO:2, wherein the cell produces a class 2:1ratio of avermectins that is different from the ratio produced by a cellof the same S. avermitilis strain which express the wild-type aveCallele.

[0148] It is a primary objective of the screening assays describedherein to identify mutated alleles of the aveC gene the expression ofwhich, in S. avermitilis cells, alters and, more particularly, reducesthe ratio of class 2:1 avermectins produced. In a preferred embodiment,the ratio of B2:B1 avermectins produced by cells of a novel S.avermitilis, strain of the present invention expressing a mutated aveCallele, or degenerate variant thereof, of the present invention is about1.6:1 or less. In a more preferred embodiment, the ratio is about 1:1 orless. In a more preferred embodiment, the ratio is about 0.84:1 or less.In a more preferred embodiment, the ratio is about 0.80:1 or less. In amore preferred embodiment, the ratio is about 0.75:1 or less. In a morepreferred embodiment, the ratio is about 0.73:1 or less. In a morepreferred embodiment, the ratio is about 0.68:1 or less. In an even morepreferred embodiment, the ratio is about 0.67:1 or less. In a morepreferred embodiment, the ratio is about 0.57:1 or less. In an even morepreferred embodiment, the ratio is about 0.53:1 or less. In an even morepreferred embodiment, the ratio is about 0.42:1 or less. In an even morepreferred embodiment, the ratio is about 0.40:1 or less.

[0149] In a specific embodiment described below, novel cells of thepresent invention produce cyclohexyl B2:cyclohexyl B1 avermectins in aratio of less than 1.6:1. In a different specific embodiment describedbelow, novel cells of the present invention produce cyclohexylB2:cyclohexyl B1 avermectins in a ratio of about 0.94:1. In a furtherdifferent specific embodiment described below, novel cells of thepresent invention produce cyclohexyl B2:cyclohexyl B1 avermectins in aratio of about 0.88:1. In a further different specific embodimentdescribed below, novel cells of the present invention produce cyclohexyl2:cyclohexyl B1 avermectins in a ratio of about 0.84:1. In a stillfurther different specific embodiment described below, novel cells ofthe present invention produce cyclohexyl 2:cyclohexyl B1 avermectins ina ratio of about 0.75:1. In a still further different specificembodiment described below, novel cells of the present invention producecyclohexyl 2:cyclohexyl B1 avermectins in a ratio of about 0.73:1. In astill further different specific embodiment described below, novel cellsof the present invention produce cyclohexyl 2:cyclohexyl B1 avermectinsin a ratio of about 0.68:1. In a still further different specificembodiment described below, novel cells of the present invention producecyclohexyl 2:cyclohexyl B1 avermectins in a ratio of about 0.67:1. In astill further different specific embodiment described below, novel cellsof the present invention produce cyclohexyl 2:cyclohexyl B1 avermectinsin a ratio of about 0.57:1. In a still further different specificembodiment described below, novel cells of the present invention producecyclohexyl 2:cyclohexyl B1 avermectins in a ratio of about 0.53:1. In astill further different specific embodiment described below, novel cellsof the present invention produce cyclohexyl 2:cyclohexyl B1 avermectinsin a ratio of about 0.42:1. In yet a further different specificembodiment described below, novel cells of the present invention producecyclohexyl 2:cyclohexyl B1 avermectins in a ratio of about 0.40:1.

[0150] In a further preferred embodiment, the present invention providesnovel strains of S. avermitilis comprising cells which express a mutatedaveC allele or a degenerate variant thereof, or a genetic constructcomprising an aveC allele or a degenerate variant thereof, in place of,or in addition to, the wild-type aveC allele, wherein the cells of thenovel strain produce an altered amount of avermectins compared to cellsof the same strain that instead express only the wild-type aveC allele.In a preferred embodiment, the novel strain produces an increased amountof avermectins. In a non-limiting embodiment, the genetic constructfurther comprises a strong promoter, such as the strong constitutiveermE promoter from Saccharopolyspora erythraea, upstream from and inoperative association with the aveC ORF.

[0151] In a further preferred embodiment, the present invention providesnovel strains of S. avermitilis comprising cells in which the aveC genehas been inactivated. Such strains are useful both for the differentspectrum of avermectins that they produce compared to the wild-typestrain, and in complementation screening assays as described herein, todetermine whether targeted or random mutagenesis of the aveC geneaffects avermectin production. In a specific embodiment described below,S. avermitilis host cells were genetically engineered to contain aninactivated aveC gene. For example, strain SE180-11, described in theexamples below, was generated using the gene replacement plasmid pSE180(ATCC 209605) (FIG. 3), which was constructed to inactivate the S.avermitilis aveC gene by insertion of the ermE resistance gene into theaveC coding region.

[0152] The present invention further provides recombinantly expressedmutated S. avermitilis AveC gene products encoded by any of theaforementioned polynucleotide molecules of the invention, and methods ofpreparing the same.

[0153] The present invention further provides a process for producingavermectins, comprising culturing cells of a strain of S. avermitilis,which cells express a mutated aveC allele that encodes a gene productthat alters the class 2:1 ratio of avermectins produced by cells of astrain of S. avermitilis expressing the mutated aveC allele compared tocells of the same strain that instead express only the wild-type aveCallele, in culture media under conditions that permit or induce theproduction of avermectins therefrom, and recovering said avermectinsfrom the culture. In a preferred embodiment, the class 2:1 ratio ofavermectins produced in the culture by cells expressing the mutated aveCallele is reduced. This process provides increased efficiency in theproduction of commercially valuable avermectins such as doramectin.

[0154] The present invention further provides a process for producingavermectins, comprising culturing cells of a strain of S. avermitilis,which cells express a mutated aveC allele or a genetic constructcomprising an aveC allele that results in the production of an alteredamount of avermectins produced by cells of a strain of S. avermitilisexpressing the mutated aveC allele or genetic construct compared tocells of the same strain which do not express the mutated aveC allele orgenetic construct but instead express only the wild-type aveC allele, inculture media under conditions that permit or induce the production ofavermectins therefrom, and recovering said avermectins from the culture.In a preferred embodiment, the amount of avermectins produced in cultureby cells expressing the mutated aveC allele, degenerate variant orgenetic construct is increased.

[0155] The present invention further provides a novel composition ofavermectins produced by a strain of S. avermitilis expressing a mutatedaveC allele or degenerate variant thereof that encodes a gene productthat reduces the class 2:1 ratio of avermectins produced by cells of astrain of S. avermitilis expressing the mutated aveC allele ordegenerate variant compared to cells of the same strain that insteadexpress only the wild-type aveC allele, wherein the avermectins in thenovel composition are produced in a reduced class 2:1 ratio as comparedto the class 2:1 ratio of avermectins produced by cells of the samestrain of S. avermitilis that instead express only the wild-type aveCallele. The novel avermectin composition can be present as produced inexhausted fermentation culture fluid, or can be harvested therefrom. Thenovel avermectin composition can be partially or substantially purifiedfrom the culture fluid by known biochemical techniques of purification,such as by ammonium sulfate precipitation, dialysis, size fractionation,ion exchange chromatography, HPLC, etc.

5.4. Uses Of Avermectins

[0156] Avermectins are highly active antiparasitic agents havingparticular utility as anthelmintics, ectoparasiticides, insecticides andacaricides. Avermectin compounds produced according to the methods ofthe present invention are useful for any of these purposes. For example,avermectin compounds produced according to the present invention areuseful to treat various diseases or conditions in humans, particularlywhere those diseases or conditions are caused by parasitic infections,as known in the art. See, e.g., Ikeda and Omura, 1997, Chem. Rev.97(7):2591-2609. More particularly, avermectin compounds producedaccording to the present invention are effective in treating a varietyof diseases or conditions caused by endoparasites, such as parasiticnematodes, which can infect humans, domestic animals, swine, sheep,poultry, horses or cattle.

[0157] More specifically, avermectin compounds produced according to thepresent invention are effective against nematodes that infect humans, aswell as those that infect various species of animals. Such nematodesinclude gastrointestinal parasites such as Ancylostoma, Necator,Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius,Dirofilaria, and parasites that are found in the blood or other tissuesor organs, such as filarial worms and the extract intestinal states ofStrongyloides and Trichinella.

[0158] The avermectin compounds produced according to the presentinvention are also useful in treating ectoparasitic infectionsincluding, e.g., arthropod infestations of mammals and birds, caused byticks, mites, lice, fleas, blowflies, biting insects, or migratingdipterous larvae that can affect cattle and horses, among others.

[0159] The avermectin compounds produced according to the presentinvention are also useful as insecticides against household pests suchas, e.g., the cockroach, clothes moth, carpet beetle and the houseflyamong others, as well as insect pests of stored grain and ofagricultural plants, which pests include spider mites, aphids,caterpillars, and orthopterans such as locusts, among others.

[0160] Animals that can be treated with the avermectin compoundsproduced according to the present invention include sheep, cattle,horses, deer, goats, swine, birds including poultry, and dogs and cats.

[0161] An avermectin compound produced according to the presentinvention is administered in a formulation appropriate to the specificintended use, the particular species of host animal being treated, andthe parasite or insect involved. For use as a parasiticide, anavermectin compound produced according to the present invention can beadministered orally in the form of a capsule, bolus, tablet or liquiddrench or, alternatively, can be administered as a pour-on, or byinjection, or as an implant Such formulations are prepared in aconventional manner in accordance with standard veterinary practice.Thus, capsules, boluses or tablets can be prepared by mixing the activeingredient with a suitable finely divided diluent or carrieradditionally containing a disintegrating agent and/or binder such asstarch, lactose, talc, magnesium stearate, etc. A drench formulation canbe prepared by dispersing the active ingredient in an aqueous solutiontogether with a dispersing or wetting agent, etc. Injectableformulations can be prepared in the form of a sterile solution, whichcan contain other substances such as, e.g., sufficient salts and/orglucose to make the solution isotonic with blood.

[0162] Such formulations will vary with regard to the weight of activecompound depending on the patient, or species of host animal to betreated, the severity and type of infection, and the body weight of thehost. Generally, for oral administration a dose of active compound offrom about 0.001 to 10 mg per kg of patient or animal body weight givenas a single dose or in divided doses for a period of from 1 to 5 dayswill be satisfactory. However, there can be instances where higher orlower dosage ranges are indicated, as determined, e.g., by a physicianor veterinarian, as based on clinical symptoms.

[0163] As an alternative, an avermectin compound produced according tothe present invention can be administered in combination with animalfeedstuff, and for this purpose a concentrated feed additive or premixcan be prepared for mixing with the normal animal feed.

[0164] For use as an insecticide, and for treating agricultural pests,an avermectin compound produced according to the present invention canbe applied as a spray, dust, emulsion and the like in accordance withstandard agricultural practice.

6. EXAMPLE: FERMENTATION OF STREPTOMYCES AVERMITILIS AND B2:B1AVERMECTIN ANALYSIS

[0165] Strains lacking both branched-chain 2-oxo acid dehydrogenase and5-O-methyltransferase activities produce no avermectins if thefermentation medium is not supplemented with fatty acids. This exampledemonstrates that in such mutants a wide range of B2:B1 ratios ofavermectins can be obtained when biosynthesis is initiated in thepresence of different fatty acids.

6.1. Materials And Methods

[0166]Streptomyces avermitilis ATCC 53692 was stored at −70° C. as awhole broth prepared in seed medium consisting of: Starch (Nadex, LaingNational)—20 g; Pharmamedia (Trader's Protein, Memphis, Tenn.)—15 g;Ardamine pH (Yeast Products Inc.)—5 g; calcium carbonate—1 g. Finalvolume was adjusted to 1 liter with tap water, pH was adjusted to 7.2,and the medium was autoclaved at 121° C. for 25 min.

[0167] Two ml of a thawed suspension of the above preparation was usedto inoculate a flask containing 50 ml of the same medium. After 48 hrsincubation at 28° C. on a rotary shaker at 180 rpm, 2 ml of the brothwas used to inoculate a flask containing 50 ml of a production mediumconsisting of: Starch—80 g; calcium carbonate—7 g; Pharmamedia—5 g;dipotassium hydrogen phosphate—1 g; magnesium sulfate—1 g; glutamicacid—0.6 g; ferrous sulfate heptahydrate—0.01 g; zinc sulfate—0.001 g;manganous sulfate—0.001 g. Final volume was adjusted to 1 liter with tapwater, pH was adjusted to 7.2, and the medium was autoclaved at 121° C.for 25 min.

[0168] Various carboxylic acid substrates (see TABLE 1) were dissolvedin methanol and added to the fermentation broth 24 hrs after inoculationto give a final concentration of 0.2 g/liter. The fermentation broth wasincubated for 14 days at 28° C., then the broth was centrifuged (2,500rpm for 2 min) and the supernatant discarded. The mycelial pellet wasextracted with acetone (15 ml), then with dichloromethane (30 ml), andthe organic phase separated, filtered, then evaporated to dryness. Theresidue was taken up in methanol (1 ml) and analyzed by HPLC with aHewlett-Packard 1090A liquid chromatograph equipped with a scanningdiode-array detector set at 240 nm. The column used was a BeckmanUltrasphere C-18, 5 μm, 4.6 mm×25 cm column maintained at 40° C.Twenty-five μl of the above methanol solution was injected onto thecolumn. Elution was performed with a linear gradient of methanol-waterfrom 80:20 to 95:5 over 40 min at 0.85/ml min. Two standardconcentrations of cyclohexyl B1 were used to calibrate the detectorresponse, and the area under the curves for B2 and B1 avermectins wasmeasured.

6.2. Results

[0169] The HPLC retention times observed for the B2 and B1 avermectins,and the 2:1 ratios, are shown in TABLE 1. TABLE 1 HPLC Retention Time(min) Ratio Substrate B2 B1 B2:B1 4-Tetrahydropyran carboxylic acid 8.114.5 0.25 Isobutyric acid 10.8 18.9 0.5 3-Furoic acid 7.6 14.6 0.62S-(+)-2-methylbutyric acid 12.8 21.6 1.0 Cyclohexanecarboxylic acid 16.926.0 1.6 3-Thiophenecarboxylic acid 8.8 16.0 1.8 Cyclopentanecarboxylicacid 14.2 23.0 2.0 3-Trifluoromethylbutyric acid 10.9 18.8 3.92-Methylpentanoic acid 14.5 24.9 4.2 Cycloheptanecarboxylic acid 18.629.0 15.0

[0170] The data presented in TABLE 1 demonstrates an extremely widerange of B2:B1 avermectin product ratios, indicating a considerabledifference in the results of dehydrative conversion of class 2 compoundsto class 1 compounds, depending on the nature of the fatty acid sidechain starter unit supplied. This indicates that changes in B2:B1 ratiosresulting from alterations to the AveC protein may be specific toparticular substrates. Consequently, screening for mutants exhibitingchanges in the B2:B1 ratio obtained with a particular substrate needs tobe done in the presence of that substrate. The subsequent examplesdescribed below use cyclohexanecarboxylic acid as the screeningsubstrate. However, this substrate is used merely to exemplify thepotential, and is not intended to limit the applicability, of thepresent invention.

7. EXAMPLE: ISOLATION OF THE aveC GENE

[0171] This example describes the isolation and characterization of aregion of the Streptomyces avermitilis chromosome that encodes the AveCgene product. As demonstrated below, the aveC gene was identified ascapable of modifying the ratio of cyclohexyl-B2 to cyclohexyl-B1 (B2:B1)avermectins produced.

7.1. Materials And Methods 7.1.1. Growth Of Streptomyces For DNAIsolation

[0172] The following method was followed for growing Streptomyces.Single colonies of S. avermitilis ATCC 31272 (single, colony isolate #2)were isolated on ½ strength YPD-6 containing: Difco Yeast Extract—5 g;Difco Bacto-peptone—5 g; dextrose—2.5 g; MOPS—5 g; Difco Bacto agar—15g. Final volume was adjusted to 1 liter with dH₂O, pH was adjusted to7.0, and the medium was autoclaved at 121° C. for 25 min.

[0173] The mycelia grown in the above medium were used to inoculate 10ml of TSB medium (Difco Tryptic Soy Broth—30 g, in 1 liter dH₂O,autoclaved at 121° C. for 25 min) in a 25 mm×150 mm tube which wasmaintained with shaking (300 rpm) at 28° C. for 48-72 hrs.

7.1.2. Chromosomal DNA Isolation From Streptomyces

[0174] Aliquots (0.25 ml or 0.5 ml) of mycelia grown as described abovewere placed in 1.5 ml microcentrifuge tubes and the cells concentratedby centrifugation at 12,000×g for 60 sec. The supernatant was discardedand the cells were resuspended in 0.25 ml TSE buffer (20 ml 1.5 Msucrose, 2.5 ml 1 M Tris-HCl, pH 8.0, 2.5 ml 1 M EDTA, pH 8.0, and 75 mldH₂O) containing 2 mg/ml lysozyme. The samples were incubated at 37° C.for 20 min with shaking, loaded into an AutoGen 540™ automated nucleicacid isolation instrument (Integrated Separation Systems, Natick,Mass.), and genomic DNA isolated using Cycle 159 (equipment software)according to manufacturers instructions.

[0175] Alternatively, 5 ml of mycelia were placed in a 17 mm×100 mmtube, the cells concentrated by centrifugation at 3,000 rpm for 5 min,and the supernatant removed. Cells were resuspended in 1 ml TSE buffer,concentrated by centrifugation at 3,000 rpm for 5 min, and thesupernatant removed. Cells were resuspended in 1 ml TSE buffercontaining 2 mg/ml lysozyme, and incubated at 37° C. with shaking for3060 min. After incubation, 0.5 ml 10% sodium dodecyl sulfate (SDS) wasadded and the cells incubated at 37° C. until lysis was complete. Thelysate was incubated at 65° C. for 10 min, cooled to rm temp, split intotwo 1.5 ml Eppendorf tubes, and extracted 1× with 0.5 mlphenol/chloroform (50% phenol previously equilibrated with 0.5 M Tris,pH 8.0; 50% chloroform). The aqueous phase was removed and extracted 2to 5× with chloroform:isoamyl alcohol (24:1). The DNA was precipitatedby adding {fraction (1/10)} volume 3M sodium acetate, pH 4.8, incubatingthe mixture on ice for 10 min, centrifuging the mixture at 15,000 rpm at5° C. for 10 min, and removing the supernatant to a clean tube to which1 volume of isopropanol was added. The supernatant plus isopropanolmixture was then incubated on ice for 20 min, centrifuged at 15,000 rpmfor 20 min at 5° C., the supernatant removed, and the DNA pellet washed1× with 70% ethanol. After the pellet was dry, the DNA was resuspendedin TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

7.1.3. Plasmid DNA Isolation From Streptomyces

[0176] An aliquot (1.0 ml) of mycelia was placed in 1.5 mlmicrocentrifuge tubes and the cells concentrated by centrifugation at12,000×g for 60 sec. The supernatant was discarded, the cells wereresuspended in 1.0 ml 10.3% sucrose and concentrated by centrifugationat 12,000×g for 60 sec, and the supernatant discarded. The cells werethen resuspended in 0.25 ml TSE buffer containing 2 mg/ml lysozyme, andincubated at 37° C. for 20 min with shaking and loaded into the AutoGen540™ automated nucleic acid isolation instrument Plasmid DNA wasisolated using Cycle 106 (equipment software) according tomanufacturer's instructions.

[0177] Alternatively, 1.5 ml of mycelia were placed in 1.5 mlmicrocentrifuge tubes and the cells concentrated by centrifugation at12,000×g for 60 sec. The supernatant was discarded, the cells wereresuspended in 1.0 ml 10.3% sucrose and concentrated by centrifugationat 12,000×g for 60 sec, and the supernatant discarded. The cells wereresuspended in 0.5 ml TSE buffer containing 2 mg/ml lysozyme, andincubated at 37° C. for 15-30 min. After incubation, 0.25 ml alkalineSDS (0.3N NaOH, 2% SDS) was added and the cells incubated at 55° C. for15-30 min or until the solution was clear. Sodium acetate (0.1 ml, 3M,pH 4.8) was added to the DNA solution, which was then incubated on icefor 10 min. The DNA samples were centrifuged at 14,000 rpm for 10 min at5° C. The supernatant was removed to a clean tube, and 0.2 mlphenol:chloroform (50% phenol:50% chloroform) was added and gentlymixed. The DNA solution was centrifuged at 14,000 rpm for 10 min at 5°C. and the upper layer removed to a clean Eppendorf tube. Isopropanol(0.75 ml) was added, and the solution was gently mixed and thenincubated at rm temp for 20 min. The DNA solution was centrifuged at14,000 rpm for 15 min at 5° C., the supernatant removed, and the DNApellet was washed with 70% ethanol, dried, and resuspended in TE buffer.

7.1.4. Plasmid DNA Isolation From E. coli

[0178] A single transformed E. coli colony was inoculated into 5 mlLuria-Bertani (LB) medium (Bacto-Tryptone—10 g, Bacto-yeast extract—5 g,and NaCl—10 g in 1 liter dH₂O, pH 7.0, autoclaved at 121° C. for 25 min,and supplemented with 100 μg/ml ampicillin). The culture was incubatedovernight, and a 1 ml aliquot placed in a 1.5 ml microcentrifuge tube.The culture samples were loaded into the AutoGen 540™ automated nucleicacid isolation instrument and plasmid DNA was isolated using Cycle 3(equipment software) according to manufacturer's instructions.

7.1.5. Preparation And Transformation Of S. avermitilis Protoplasts

[0179] Single colonies of S. avermitilis were isolated on ½ strengthYPD6. The mycelia were used to inoculate 10 ml of TSB medium in a 25mm×150 mm tube, which was then incubated with shaking (300 rpm) at 28°C. for 48 hrs. One ml of mycelia was used to inoculate 50 ml YEMEmedium. YEME medium contains per liter. Difco Yeast Extract—3 g; DifcoBacto-peptone—5 g; Difco Malt Extract—3 g; Sucrose—300 g. Afterautoclaving at 121° C. for 25 min, the following were added: 2.5 MMgCl₂·6H₂O (separately autoclaved at 121° C. for 25 min)—2 ml; andglycine (20%) (filter-sterilized)—25 ml.

[0180] The mycelia were grown at 30° C. for 48-72 hrs and harvested bycentrifugation in a 50 ml centrifuge tube (Falcon) at 3,000 rpm for 20min. The supernatant was discarded and the mycelia were resuspended in Pbuffer, which contains: sucrose—205 g; K₂SO₄—0.25 g; MgCl₂·6H₂O—2.02 g;H₂O—600 ml; K₂PO₄ (0.5%)—10 ml; trace element solution*—20 ml;CaCl₂·2H₂O (3.68%)—100 ml; and MES buffer (1.0 M, pH 6.5)—10 ml. (*Traceelement solution contains per liter. ZnCl₂—40 mg; FeCl₃·6H₂O—200 mg;CuCl₂·2H₂O—10 mg; MnCl₂·4H₂O—10 mg; Na₂B₄O₇·10H₂O—10 mg; (NH₄)₆Mo₇O₂₄·4H₂O—10 mg). The pH was adjusted to 6.5, final volume wasadjusted to 1 liter, and the medium was filtered hot through a 0.45micron filter.

[0181] The mycelia were pelleted at 3,000 rpm for 20 min, thesupernatant was discarded, and the mycelia were resuspended in 20 ml Pbuffer containing 2 mg/ml lysozyme. The mycelia were incubated at 35° C.for 15 min with shaking, and checked microscopically to determine extentof protoplast formation. When protoplast formation was complete, theprotoplasts were centrifuged at 8,000 rpm for 10 min. The supernatantwas removed and the protoplasts were resuspended in 10 ml P buffer. Theprotoplasts were centrifuged at 8,000 rpm for 10 min, the supernatantwas removed, the protoplasts were resuspended in 2 ml P buffer, andapproximately 1×10⁹ protoplasts were distributed to 2.0 ml cryogenicvials (Nalgene).

[0182] A vial containing 1×10⁹ protoplasts was centrifuged at 8,000 rpmfor 10 min, the supernatant was removed, and the protoplasts wereresuspended in 0.1 ml P buffer. Two to 5 μg of transforming DNA wereadded to the protoplasts, immediately followed by the addition of 0.5 mlworking T buffer. T buffer base contains: PEG-1000 (Sigma)—25 g;sucrose—2.5 g; H₂O—83 ml. The pH was adjusted to 8.8 with 1 N NaOH(filter sterilized), and the T buffer base was filter-sterilized andstored at 4° C. Working T buffer, made the same day used, was T bufferbase—8.3 ml; K₂PO₄ (4 mM)—1.0 ml; CaCl₂·2H₂O (5 M)—0.2 ml; and TES (1 M,pH 8)—0.5 ml. Each component of the working T buffer was individuallyfilter-sterilized.

[0183] Within 20 sec of adding T buffer to the protoplasts, 1.0 ml Pbuffer was also added and the protoplasts were centrifuged at 8,000 rpmfor 10 min. The supernatant was discarded and the protoplasts wereresuspended in 0.1 ml P buffer. The protoplasts were then plated on RM14media, which contains: sucrose—205 g; K₂SO₄—0.25 g; MgCl₂·6H₂O—10.12 g;glucose—10 g; Difco Casamino Acids—0.1 g; Difco Yeast Extract—5 g; DifcoOatmeal Agar—3 g; Difco Bacto Agar—22 g; dH₂O—800 ml. The solution wasautoclaved at 121° C. for 25 min. After autoclaving, sterile stocks ofthe following were added: K₂PO4 (0.5%)—10 ml; CaCl₂·2H₂O (5 M)—5 ml;L-proline (20%)—15 ml; MES buffer (1.0 M, pH 6.5)—10 ml; trace elementsolution (same as above)—2 ml; cycloheximide stock (25 mg/ml)—40 ml; and1N NaOH—2 ml. Twenty-five ml of RM14 medium were aliquoted per plate,and plates dried for 24 hr before use.

[0184] The protoplasts were incubated in 95% humidity at 30° C. for20-24 hrs. To select thiostrepton resistant transformants, 1 ml ofoverlay buffer containing 125 μg per ml thiostrepton was spread evenlyover the RM14 regeneration plates. Overlay buffer contains per 100 ml:sucrose—10.3 g; trace element solution (same as above)—0.2 ml; and MES(1 M, pH 6.5)—1 ml. The protoplasts were incubated in 95% humidity at30° C. for 7-14 days until thiostrepton resistant (Thio^(r)) colonieswere visible.

7.1.6. Transformation Of Streptomyces lividans Protoplasts

[0185]S. lividans TK64 (provided by the John Innes Institute, Norwich,U.K) was used for transformations in some cases. Methods andcompositions for growing, protoplasting, and transforming S. lividansare described in Hopwood et al., 1985, Genetic Manipulation ofStreptomyces, A Laboratory Manual, John Innes Foundation, Norwich, U.K,and performed as described therein. Plasmid DNA was isolated from S.lividans transformants as described in Section 7.1.3, above.

7.1.7. Fermentation Analysis Of S. avermitilis Strains

[0186]S. avermitilis mycelia grown on 112 strength YPD6 for 4-7 dayswere inoculated into 1×6 inch tubes containing 8 ml of preform mediumand two 5 mm glass beads. Preform medium contains: soluble starch(either thin boiled starch or KOSO, Japan Corn Starch Co., Nagoya)—20g/L, Pharmamedia—15 g/L; Ardamine pH—5 g/L (Champlain Ind., Clifton,N.J.); CaCO₃—2 g/L; 2× bcfa (“bcfa” refers to branched chain fattyacids) containing a final concentration in the medium of 50 ppm2-(+/−)-methyl butyric acid, 60 ppm isobutyric acid, and 20 ppmisovaleric acid. The pH was adjusted to 7.2, and the medium wasautoclaved at 121° C. for 25 min.

[0187] The tube was shaken at a 17° angle at 215 rpm at 29° C. for 3days. A 2-ml aliquot of the seed culture was used to inoculate a 300 mlErlenmeyer flask containing 25 ml of production medium which contains:starch (either thin boiled starch or KOSO)—160 g/L; Nutrisoy (ArcherDaniels Midland, Decatur, Ill.)—10 g/L; Ardamine pH—10 g/L; K₂HPO₄—2g/L; MgSO₄.4H₂O—2 g/L; FeSO₄.7H₂O—0.02 g/L; MnCl₂—0.002 g/L;ZnSO₄.7H₂O—0.002 g/L; CaCO₃—14 g/L; 2× bcfa (as above); and cyclohexanecarboxylic acid (CHC) (made up as a 20% solution at pH 7.0)—800 ppm. ThepH was adjusted to 6.9, and the medium was autoclaved at 121° C. for 25min.

[0188] After inoculation, the flask was incubated at 29° C. for 12 dayswith shaking at 200 rpm. After incubation, a 2 ml sample was withdrawnfrom the flask, diluted with 8 ml of methanol, mixed, and the mixturecentrifuged at 1,250×g for 10 min to pellet debris. The supernatant wasthen assayed by HPLC using a Beckman Ultrasphere ODS column (25 cm×4.6mm ID) with a flow rate of 0.75 ml/min and detection by absorbance at240 nm. The mobile phase was 86/8.9/5.1 methanol/water/acetonitrile.

7.1.8. Isolation Of S. avermitilis PKS Genes

[0189] A cosmid library of S. avermitilis (ATCC 31272, SC-2) chromosomalDNA was prepared and hybridized with a ketosynthase (KS) probe made froma fragment of the Saccharopolyspora erythraea polyketide synthase (PKS)gene. A detailed description of the preparation of cosmid libraries canbe found in Sambrook et al., 1989, above. A detailed description of thepreparation of Streptomyces chromosomal DNA libraries is presented inHopwood et al., 1985, above. Cosmid clones containingketosynthase-hybridizing regions were identified by hybridiation to a2.7 Kb Ndel/Eco47III fragment from pEX26 (kindly supplied by Dr. P.Leadlay, Cambridge, UK). Approximately 5 ng of pEX26 were digested usingNdel and Eco47III. The reaction mixture was loaded on a 0.8% SeaPlaqueGTG agarose gel (FMC BioProducts, Rockland, Me.). The 2.7 KbNdel/Eco47III fragment was excised from the gel after electrophoresisand the DNA recovered from the gel using GELase™ from EpicentreTechnologies using the Fast Protocol. The 2.7 Kb Ndel/Eco47III fragmentwas labeled with [α-³²P]dCTP (deoxycytidine 5′-triphosphate, tetra(triethylammonium) salt, [alpha-³²P]-) (NEN-Dupont, Boston, Mass.) usingthe BRL Nick Translation System (BRL Life Technologies, Inc.,Gaithersburg, Md.) following the supplier's instructions. A typicalreaction was performed in 0.05 ml volume. After addition of 5 μl Stopbuffer, the labeled DNA was separated from unincorporated nucleotidesusing a G-25 Sephadex Quick Spin™ Column (Boehringer Mannheim) followingsupplier's instructions.

[0190] Approximately 1,800 cosmid clones were screened by colonyhybridization. Ten clones were identified that hybridized strongly tothe Sacc. erythraea KS probe. E coli colonies containing cosmid DNA weregrown in LB liquid medium and cosmid DNA was isolated from each culturein the AutoGen 540™ automated nucleic acid isolation instrument usingCycle 3 (equipment software) according to manufacturer's instructions.Restriction endonuclease mapping and Southern blot hybridizationanalyses revealed that five of the clones contained overlappingchromosomal regions. An S. avermitilis genomic BamHI restriction map ofthe five cosmids (i.e., pSE65, pSE66, pSE67, pSE68, pSE69) wasconstructed by analysis of overlapping cosmids and hybridizations (FIG.4).

7.1.9. Identification Of DNA Thai Modulates Avermectin B2:B1 Ratios AndIdentification Of An aveC ORF

[0191] The following methods were used to test subcloned fragmentsderived from the pSE66 cosmid clone for their ability to modulateavermectin B2:B1 ratios in AveC mutants. pSE66 (5 μg) was digested withSad and BamHI. The reaction mixture was loaded on a 0.8% SeaPlaque™ GTGagarose gel (FMC BioProducts), a 2.9 Kb Sacl/BamHI fragment was excisedfrom the gel after electrophoresis, and the DNA was recovered from thegel using GELase™ (Epicentre Technologies) using the Fast Protocol.Approximately 5 μg of the shuttle vector pWHM3 (Vara et al., 1989, J.Bacteriol. 171:5872-5881) was digested with Sacl and BamHI. About 0.5 μgof the 2.9 Kb insert and 0.5 μg of digested pWHM3 were mixed togetherand incubated overnight with 1 unit of ligase (New England Biolabs,Inc., Beverly, Mass.) at 15° C., in a total volume of 20 μl, accordingto supplier's instructions. After incubation, 5 μl of the ligationmixture was incubated at 70° C. for 10 min, cooled to rm temp, and usedto transform competent E. coli DH5α cells (BRL) according tomanufacturer's instructions. Plasmid DNA was isolated from ampicillinresistant transformants and the presence of the 2.9 Kb Sacl/BamHI insertwas confirmed by restriction analysis. This plasmid was designated aspSE119.

[0192] Protoplasts of S. avermitilis strain 1100-SC38 (Pfizer in-housestrain) were prepared and transformed with pSE119 as described inSection 7.1.5 above. Strain 1100-SC38 is a mutant that producessignificantly more of the avermectin cyclohexyl-B2 form compared toavermectin cyclohexyl-B1 form when supplemented with cyclohexanecarboxylic acid (B2:B1 of about 30:1). pSE119 used to transform S.avermitilis protoplasts was isolated from either E. coli strain GM2163(obtained from Dr. B. J. Bachmann, Curator, E. coli Genetic StockCenter, Yale University), E. coli strain DM1 (BRL), or S. lividansstrain TK64. Thiostrepton resistant transformants of strain 1100C38 wereisolated and analyzed by HPLC analysis of fermentation products.Transformants of S. avermitilis strain 1100SC38 containing pSE119produced an altered ratio of avermectin cyclohexyl-B2:cyclohexyl-B1 ofabout 3.7:1 (TABLE 2).

[0193] Having established that pSE119 was able to modulate avermectinB2:B1 ratios in an AveC mutant, the insert DNA was sequenced.Approximately 10 μg of pSE119 were isolated using a plasmid DNAisolation kit (Qiagen, Valencia, Calif.) following manufacturer'sinstructions, and sequenced using an ABI 373A Automated DNA Sequencer(Perkin Elmer, Foster City, Calif.). Sequence data was assembled andedited using Genetic Computer Group programs (GCG, Madison, Wis.). TheDNA sequence and the aveC ORF are presented in FIG. 1 (SEQ ID NO:1).

[0194] A new plasmid, designated as pSE118, was constructed as follows.Approximately 5 μg of pSE66 was digested with Sphl and BamHI. Thereaction mixture was loaded on a 0.8% SeaPlaque GTG agarose gel (FMCBioProducts), a 2.8 Kb Sphl/BamHI fragment was excised from the gelafter electophoresis, and the DNA was recovered from the gel usingGELase™ (Epicentre Technologies) using the Fast Protocol. Approximately5 μg of the shuttle vector pWHM3 was digested with Sphl and BamHI. About0.5 μg of the 2.8 Kb insert and 0.5 μg of digested pWHM3 were mixedtogether and incubated overnight with I unit of ligase (New EnglandBiolabs) at 15° C. in a total volume of 20 μl according to supplier'sinstructions. After incubation, 5 μl of the ligation mixture wasincubated at 70° C. for 10 min, cooled to rm temp, and used to transformcompetent E. coli DH5α cells according to manufacturer's instructions.Plasmid DNA was isolated from ampicillin resistant transformants, andthe presence of the 2.8 Kb Sphl/BamHI insert was confirmed byrestriction analysis. This plasmid was designated as pSE118. The insertDNA in pSE118 and pSE119 overlap by approximately 838 nucleotides (FIG.4).

[0195] Protoplasts of S. avermitilis strain 1100-SC38 were transformedwith pSE118 as above. Thiostrepton resistant transformants of strain1100-SC38 were isolated and analyzed by HPLC analysis of fermentationproducts. Transformants of S. avermitilis strain I 100-SC38 containingpSE118 were not altered in the ratios of avermectin cyclohexyl-B2:avermectin cyclohexyl-B1 compared to strain 1100SC38 (TABLE 2).

7.1.10. PCR Amplification Of The aveC Gene From S. avermitilisChromosomal DNA

[0196] A ˜1.2 Kb fragment containing the aveC ORF was isolated from S.avermitilis chromosomal DNA by PCR amplification using primers designedon the basis of the aveC nucleotide sequence obtained above. The PCRprimers were supplied by Genosys Biotechnologies, Inc. (Tex.). Therightward primer was: 5′-TCACGAAACCGGACACAC-3′ (SEQ ID NO:6); and theleftward primer was: 5′-CATGATCGCTGAACCGAG3′ (SEQ ID NO:7). The PCRreaction was carried out with Deep Vent™ polymerase (New EnglandBiolabs) in buffer provided by the manufacturer, and in the presence of300 μM dNTP, 10% glycerol, 200 pmol of each primer, 0.1 μg template, and2.5 units enzyme in a final volume of 100 μl, using a Perkin-Elmer Cetusthermal cycler. The thermal profile of the first cycle was 95° C. for 5min (denaturation step), 60° C. for 2 min (annealing step), and 72° C.for 2 min (extension step). The subsequent 24 cycles had a similarthermal profile except that the denaturation step was shortened to 45sec and the annealing step was shortened to I min.

[0197] The PCR product was electrophoresed in a 1% agarose gel and asingle DNA band of ˜1.2 Kb was detected. This DNA was purified from thegel, and ligated with 25 ng of linearized, blunt pCR-Blunt vector(Invitrogen) in a 1:10 molar vector-to-insert ratio followingmanufacturer's instructions. The ligation mixture was used to transformOne Shot™ Competent E. coli cells (Invitrogen) following manufacturer′sinstructions. Plasmid DNA was isolated from ampicillin resistanttransformants, and the presence of the -1.2 Kb insert was confirmed byrestriction analysis. This plasmid was designated as pSE179.

[0198] The insert DNA from pSE179 was isolated by digestion withBamHI/Xbal, separated by electrophoresis, purified from the gel, andligated with shuttle vector pWHM3, which had also been digested withBamHI/Xbal, in a total DNA concentration of 1 μg in a 1:5 molarvector-to-insert ratio. The ligation mixture was used to transformcompetent E. coli DH5α cells according to manufacturer's instructions.Plasmid DNA was isolated from ampicillin resistant transformants and thepresence of the ˜1.2 Kb insert was confirmed by restriction analysis.This plasmid, which was designated as pSE186 (FIG. 2, ATCC 209604), wastransformed into E. coli DM1, and plasmid DNA was isolated fromampicillin resistant transformants.

7.2. Results

[0199] A 2.9 Kb Sacl/BamHI fragment from pSE119 was identified that,when transformed into S. avermitilis strain 1100-SC38, significantlyaltered the ratio of B2:B1 avermectin production. S. avermitilis strain1100-SC38 normally has a B2:B1 ratio of about 30:1, but when transformedwith a vector comprising the 2.9 Kb Sacl/BamHI fragment, the ratio ofB2:B1 avermectin decreased to about 3.7:1. Post-fermentation analysis oftransformant cultures verified the presence of the transforming DNA

[0200] The 2.9 Kb pSE119 fragment was sequenced and a 0.9 Kb ORF wasidentified (FIG. 1) (SEQ ID NO:1), which encompasses a Pstl/Sphlfragment that had previously been mutated elsewhere to produce B2products only (Ikeda et al., 1995, above). A comparison of this ORF, orits corresponding deduced polypeptide, against known databases (GenEMBL,SWISS-PROT) did not show any strong homology with known DNA or proteinsequences.

[0201] TABLE 2 presents the fermentation analysis of S. avermitilisstrain 1100-SC38 transformed with various plasmids. TABLE 2 Avg. S.avermitilis strain No. Transformants B2:B1 (transforming plasmid) TestedRatio 1100-SC38 (none)  9 30.66 1100-SC38 (pWHM3) 21 31.3 1100-SC38(pSE119) 12 3.7 1100-SC38 (pSE118) 12 30.4 1100-SC38 (pSE185) 14 27.9

8. EXAMPLE: CONSTRUCTION OF S. AVERMITILIS AveC MUTANTS

[0202] This example describes the construction of several different S.avermitilis AveC mutants using the compositions and methods describedabove. A general description of techniques for introducing mutationsinto a gene in Streptomyces is described by Kieser and Hopwood, 1991,Meth. Enzym. 204:430458. A more detailed description is provided byAnzai et al., 1988, J. Antibiot. XLI(2):226-233, and by Stutzman-Engwallet al., 1992, J. Bacteriol. 174(1):144-154. These references areincorporated herein by reference in their entirety.

8.1. Inactivation Of The S. avermitilis aveC Gene

[0203] AveC mutants containing inactivated aveC genes were constructedusing several methods, as detailed below.

[0204] In the first method, a 640 bp Sphl/Pstl fragment internal to theaveC gene in pSE119 (plasmid described in Section 7.1.9, above) wasreplaced with the ermE gene (for erythromycin resistance) from Sacc.erythraea. The ermE gene was isolated from plJ4026 (provided by the JohnInnes Institute, Norwich, U.K; see also Bibb et al., 1985, Gene41:357-368) by restriction enzyme digestion with Bg/II and EcoRI,followed by electrophoresis, and was purified from the gel. This ˜1.7 Kbfragment was ligated into pGEM7Zf (Promega) which had been digested withBamHI and EcoRI, and the ligation mixture transformed into competent E.coli DH5α cells following manufacturer's instructions. Plasmid DNA wasisolated from ampicillin resistant transformants, and the presence ofthe ˜1.7 Kb insert was confirmed by restriction analysis. This plasmidwas designated as pSE27.

[0205] pSE118 (described in Section 7.1.9, above) was digested with Sphland BamHI, the digest electrophoresed, and the ˜2.8 Kb Sphl/BamHI insertpurified from the gel. pSE119 was digested with Pstl and EcoRI, thedigest electrophoresed, and the —1.5 Kb Pstl/EcoRI insert purified fromthe gel. Shuttle vector pWHM3 was digested with BamHI and EcoRI. pSE27was digested with Pstl and Sphl, the digest electrophoresed, and the˜1.7 Kb Pstl/Sphl insert purified from the gel. All four fragments(i.e., ˜2.8 Kb, ˜1.5Kb, ˜7.2Kb, ˜1.7Kb) were ligated together in a 4-wayligation. The ligation mixture was transformed into competent E. coliDH5α cells following manufacturer's instructions. Plasmid DNA wasisolated from ampicillin resistant transformants, and the presence ofthe correct insert was confirmed by restriction analysis. This plasmidwas designated as pSE180 (FIG. 3; ATCC 209605).

[0206] pSE180 was transformed into S. lividans TK64 and transformedcolonies identified by resistance to thiostrepton and erythromycin.pSE180 was isolated from S. lividans and used to transform S.avermitilis protoplasts. Four thiostrepton resistant S. avermitilistransformants were identified, and protoplasts were prepared and platedunder non-selective conditions on RM14 media. After the protoplasts hadregenerated, single colonies were screened for the presence oferythromycin resistance and the absence of thiostrepton resistance,indicating chromosomal integration of the inactivated aveC gene and lossof the free replicon. One Erm^(r) Thio^(r) transformant was identifiedand designated as strain SE180-11. Total chromosomal DNA was isolatedfrom strain SE180-11, digested with restriction enzymes BamHI, HindIII,Pstl, or Sphl, resolved by electrophoresis on a 0.8% agarose gel,transferred to nylon membranes, and hybridized to the ermE probe. Theseanalyses showed that chromosomal integration of the ermE resistancegene, and concomitant deletion of the 640 bp Pstl/Sphl fragment hadoccurred by a double crossover event. HPLC analysis of fermentationproducts of strain SE180-11 showed that normal avermectins were nolonger produced (FIG. 5A).

[0207] In a second method for inactivating the aveC gene, the 1.7 KbermE gene was removed from the chromosome of S. avermitilis strainSE180-11, leaving a 640 bp Pstl/Sphl deletion in the aveC gene. A genereplacement plasmid was constructed as follows: pSE180 was partiallydigested with Xbal and an ˜11.4 Kb fragment purified from the gel. The˜11:4 Kb band lacks the 1.7 Kb ermE resistance gene. The DNA was thenligated and transformed into E. coli DH5α cells. Plasmid DNA wasisolated from ampicillin resistant transformants and the presence of thecorrect insert was confirmed by restriction analysis. This plasmid,which was designated as pSE184, was transformed into E. coli DM1, andplasmid DNA isolated from ampicillin resistant transformants. Thisplasmid was used to transform protoplasts of S. avermitilis strainSE180-11. Protoplasts were prepared from thiostrepton resistanttransformants of strain SE180-11 and were plated as single colonies onRM14. After the protoplasts had regenerated, single colonies werescreened for the absence of both erythromycin resistance andthiostrepton resistance, indicating chromosomal integration of theinactivated aveC gene and loss of the free replicon containing the ermEgene. One Erm^(r) Thio^(r) transformant was identified and designated asSE184-1-13. Fermentation analysis of SE184-1-13 showed that normalavermectins were not produced and that SE184-1-13 had the samefermentation profile as SE180-11.

[0208] In a third method for inactivating the aveC gene, a frameshiftwas introduced into the chromosomal aveC gene by adding two G's afterthe C at nt position 471 using PCR, thereby creating a BspE1 site. Thepresence of the engineered BspE1 site was useful in detecting the genereplacement event The PCR primers were designed to introduce aframeshift mutation into the aveC gene, and were supplied by GenosysBibtechnologies, Inc. The rightward primer was: 5′GGTTCCGGATGCCGTrCTCG3′(SEQ ID NO:8) and the leftward primer was: 5′-AACTCCGGTCGACTCCCCTTC-3′(SEQ ID NO:9). The PCR conditions were as described in Section 7.1.10above. The 666 bp PCR product was digested with Sphl to give twofragments of 278 bp and 388 bp, respectively. The 388 bp fragment waspurified from the gel.

[0209] The gene replacement plasmid was constructed as follows: shuttlevector pWHM3 was digested with EcoRI and BamHI. pSE119 was digested withBamHI and Sphl, the digest electrophoresed, and a ˜840 bp fragment waspurified from the gel. pSE119 was digested with EcoRI and Xmnl, thedigest was resolved by electrophoresis, and a ˜1.7 Kb fragment waspurified from the gel. All four fragments (i.e., ˜7.2 Kb, ˜840 bp, ˜1.7Kb, and 388 bp) were ligated together in a 4-way ligation. The ligationmixture was transformed into competent E. coli DH5α cells. Plasmid DNAwas isolated from ampicillin resistant transformants and the presence ofthe correct insert was confirmed by restriction analysis and DNAsequence analysis. This plasmid, which was designated as pSE185, wastransformed into E. coli DM1 and plasmid DNA isolated from ampicillinresistant transformants. This plasmid was used to transform protoplastsof S. avermitilis strain 1100-SC38. Thiostrepton resistant transformantsof strain 1100-SC38 were isolated and analyzed by HPLC analysis offermentation products. pSE185 did not significantly alter the B2:B1avermectin ratios when transformed into S. avermitilis strain 1100SC38(TABLE 2).

[0210] pSE185 was used to transform protoplasts of S. avermitilis togenerate a frameshift mutation in the chromosomal aveC gene. Protoplastswere prepared from thiostrepton resistant transformants and plated assingle colonies on RM14. After the protoplasts had regenerated, singlecolonies were screened for the absence of thiostrepton resistance.Chromosomal DNA from thiostrepton sensitive colonies was isolated andscreened by PCR for the presence of the frameshift mutation integratedinto the chromosome. The PCR primers were designed based on the aveCnucleotide sequence, and were supplied by Genosys Biotechnologies, Inc.(Tex.). The rightward PCR primer was: 5′-GCAAGGATACGGGGACTAC-3′ (SEQ IDNO:10) and the leftward PCR primer was: 5′-GAACCGACCGCCTGATAC-3′ (SEQ IDNO:11), and the PCR conditions were as described in Section 7.1.10above. The PCR product obtained was 543 bp and, when digested withBspE1, three fragments of 368 bp, 96 bp, and 79 bp were observed,indicating chromosomal integration of the inactivated aveC gene and lossof the free replicon.

[0211] Fermentation analysis of S. avermitilis mutants containing theframeshift mutation in the aveC gene showed that normal avermectins wereno longer produced, and that these mutants had the same fermentationHPLC profile as strains SE180-11 and SE184-1-13. One Thio* transformantwas identified and designated as strain SE185-5a.

[0212] Additionally, a mutation in the aveC gene that changes ntposition 520 from G to A, which results in changing the codon encoding atryptophan (W) at position 116 to a termination codon, was produced. AnS. avermitilis strain with this mutation did not produce normalavermectins and had the same fermentation profile as strains SE180-11,SE184-1-13, and SE185-5a.

[0213] Additionally, mutations in the aveC gene that change both: (i) ntposition 970 from G to A, which changes the amino acid at position 266from a glycine (G) to an aspartate (D), and (ii) nt position 996 from Tto C, which changes the amino acid at position 275 from tyrosine (Y) tohistidine (H), were produced. An S. avermitilis strain with thesemutations (G256D/Y275H) did not produce normal avermectins and had thesame fermentation profile as strains SE180-11, SE184-1-13, and SE185-5a.

[0214] The S. avermitilis aveC inactivation mutant strains SE180-11,SE184-1-13, SE185-5a, and others provided herewith, provide screeningtools to assess the impact of other mutations in the aveC gene. pSE186,which contains a wild-type copy of the aveC gene, was transformed intoE. coli DM1, and plasmid DNA was isolated from ampicillin resistanttransformants. This pSE186 DNA was used to transform protoplasts of S.avermitilis strain SE180-11. Thiostrepton resistant transformants ofstrain SE180-11 were isolated, the presence of erythromycin resistancewas determined, and Thio^(r) Erm^(r) transformants were analyzed by HPLCanalysis of fermentation products. The presence of the functional aveCgene in trans was able to restore normal avermectin production to strainSE180-11 (FIG. 5B).

8.2. Analysis Of Mutations In The aveC Gene That Alter Class B2:B1Ratios

[0215] As described above, S. avermitilis strain SE188-11 containing aninactive aveC gene was complemented by transformation with a plasmidcontaining a functional aveC gene (pSE186). Strain SE180-11 was alsoutilized as a host strain to characterize other mutations in the aveCgene, as described below.

[0216] Chromosomal DNA was isolated from strain 1100SC38, and used as atemplate for PCR amplification of the aveC gene. The 1.2 Kb ORF wasisolated by PCR amplification using primers designed on the basis of theaveC nucleotide sequence. The rightward primer was SEQ ID NO:6 and theleftward primer was SEQ ID NO:7 (see Section 7.1.10, above). The PCR andsubcloning conditions were as described in Section 7.1.10. DNA sequenceanalysis of the 1.2 Kb ORF shows a mutation in the aveC gene thatchanges nt position 337 from C to T, which changes the amino acid atposition 55 from serine (S) to phenylalanine (F). The aveC genecontaining the S55F mutation was subcloned into pWHM3 to produce aplasmid which was designated as pSE187, and which was used to transformprotoplasts of S. avermitilis strain SE180-11. Thiostrepton resistanttransformants of strain SE180-11 were isolated, the presence oferythromycin resistance was determined, and Thio^(r) Erm^(r)transformants were analyzed by HPLC analysis of fermentation products.The presence of the aveC gene encoding a change at amino acid residue 55(S55F) was able to restore normal avermectin production to strainSE180-11 (FIG. 5C); however, the cyclohexyl B2:cyclohexyl B1 ratio wasabout 26:1, as compared to strain SE180-11 transformed with pSE186,which had a ratio of B2:B1 of about 1.6:1 (TABLE 3), indicating that thesingle mutation (S55F) modulates the amount of cyclohexyl-B2 producedrelative to cyclohexyl-B1.

[0217] Another mutation in the aveC gene was identified that changes ntposition 862 from G to A, which changes the amino acid at position 230from glycine (G) to aspartate (D). An S. avermitilis strain having thismutation (G230D) produces avermectins at a B2:B1 ratio of about 30:1.

8.3 Mutations That Reduce The B2:B1 Ratio

[0218] Several mutations were constructed that reduce the amount ofcyclohexyl-B2 produced relative to cyclohexyl-B1, as follows.

[0219] A mutation in the aveC gene was identified that changes ntposition 588 from G to A, which changes the amino acid at position 139from alanine (A) to threonine (T). The aveC gene containing the A139Tmutation was subcloned into pWHM3 to produce a plasmid which wasdesignated pSE188, and which was used to transform protoplasts of S.avermitilis strain SE180-11. Thiostrepton resistant transformants ofstrain SE180-11 were isolated, the presence of erythromycin resistancewas determined, and Thio^(r) Erm^(r) transformants were analyzed by HPLCanalysis of fermentation products. The presence of the mutated aveC geneencoding a change at amino acid residue 139 (A139T) was able to restoreavermectin production to strain SE180-11 (FIG. 5D); however, the B2:B1ratio was about 0.94:1, indicating that this mutation reduces the amountof cyclohexyl-B2 produced relative to cyclohexyl-B1. This result wasunexpected because published results, as well as the results ofmutations described above, have only demonstrated either inactivation ofthe aveC gene or increased production of the B2 form of avermectinrelative to the B1 form (TABLE 3).

[0220] Because the A139T mutation altered the B2:B1 ratios in the morefavorable B1 direction, a mutation was constructed that encoded athreonine instead of a serine at amino acid position 138. Thus, pSE186was digested with EcoRI and cloned into pGEM3Zf (Promega) which had beendigested with EcoRI. This plasmid, which was designated as pSE186a, wasdigested with Apal and Kpnl, the DNA fragments separated on an agarosegel, and two fragments of ˜3.8 Kb and ˜0.4 Kb were purified from thegel. The ˜1.2 Kb insert DNA from pSE186 was used as a PCR template tointroduce a single base change at nt position 585. The PCR primers weredesigned to introduce a mutation at nt position 585, and were suppliedby Genosys Biotechnologies, Inc. (Tex.). The rightward PCR primer was:5′-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCCCTGGCGACG-3′ (SEQ ID NO: 12); andthe leftward PCR primer was: 5′-GGAACCGACCGCCTGATACA-3′ (SEQ ID NO:13).The PCR reaction was carried out using an Advantage GC genomic PCR kit(Clonetech Laboratories, Palo Alto, Calif.) in buffer provided by themanufacturer in the presence of 200 μM dNTPs, 200 pmol of each primer,50 ng template DNA, 1.0 M GC-Melt and 1 unit KlenTaq Polymerase Mix in afinal volume of 50 μl. The thermal profile of the first cycle was 94° C.for 1 min; followed by 25 cycles of 94° C. for 30 sec and 68° C. for 2min; and 1 cycle at 68° C. for 3 min. A PCR product of 295 bp wasdigested with Apal and Kpnl to release a 254 bp fragment, which wasresolved by electrophoresis and purified from the gel. All threefragments (˜3.8 Kb, ˜0.4 Kb and 254 bp) were ligated together in a 3-wayligation. The ligation mixture was transformed into competent E. coliDH5α cells. Plasmid DNA was isolated from ampicillin resistanttransformants, and the presence of the correct insert was confirmed byrestriction analysis. This plasmid was designated as pSE198.

[0221] pSE198 was digested with EcoRI, cloned into pWHM3, which had beendigested with EcoRI, and transformed into E. coli DH5α cells. PlasmidDNA was isolated from ampicillin resistant transformants and thepresence of the correct insert was confirmed by restriction analysis andDNA sequence analysis. This plasmid DNA was transformed into E. coliDM1, plasmid DNA was isolated from ampicillin resistant transformants,and the presence of the correct insert was confirmed by restrictionanatysis. This plasmid, which was designated as pSE199, was used totransform protoplasts of S. avermitilis strain SE180-11. Thiostreptonresistant transformants of strain SE180-11 were isolated, the presenceof erythromycin resistance was determined, and Thio^(r) Erm^(r)transformants were analyzed by HPLC analysis of fermentation products.The presence of the mutated aveC gene encoding a change at amino acidresidue 138 (S138T) was able to restore normal avermectin production tostrain SE180-11; however, the B2:B1 ratio was 0.88:1 indicating thatthis mutation reduces the amount of cyclohexyl-B2 produced relative tocyclohexyl-B1 (TABLE 3). This B2:B1 ratio is even lower than the 0.94:1ratio observed with the A139T mutation produced by transformation ofstrain SE180-11 with pSE188, as described above.

[0222] Another mutation was constructed to introduce a threonine at bothamino acid positions 138 and 139. The ˜1.2 Kb insert DNA from pSE186 wasused as a PCR template. The PCR primers were designed to introducemutations at nt positions 585 and 588, and were supplied by GenosysBiotechnologies, Inc. (Tex.). The rightward PCR primer was:5′-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGACC-3′ (SEQ ID NO:14); andthe leftward PCR primer was: 5′-GGAACATCACGGCATTCACC-3′ (SEQ ID NO:15).The PCR reaction was performed using the conditions describedimmediately above in this Section. A PCR product of 449 bp was digestedwith Apal and Kpnl to release a 254 bp fragment, which was resolved byelectrophoresis and purified from the gel. pSE186a was digested withApal and Kpnl, the DNA fragments separated on an agarose gel, and twofragments of ˜3.8 Kb and ˜0.4 Kb were purified from the gel. All threefragments (˜3.8 Kb, ˜0.4 Kb and 254 bp) were ligated together in a 3-wayligation, and the ligation mixture was transformed into competent E.coli DH5α cells. Plasmid DNA was isolated from ampicillin resistanttransformants, and the presence of the correct insert was confirmed byrestriction analysis. This plasmid was designated as pSE230.

[0223] pSE230 was digested with EcoRI, cloned into pWHM3, which had beendigested with EcoRI, and transformed into E. coli DH5α cells. PlasmidDNA was isolated from ampicillin resistant transformants and thepresence of the correct insert was confirmed by restriction analysis andDNA sequence analysis. This plasmid DNA was transformed into E. coliDM1, plasmid DNA isolated from ampicillin resistant transformants, andthe presence of the correct insert was confirmed by restrictionanalysis. This plasmid, which was designated as pSE231, was used totransform protoplasts of S. avermitilis strain SE180-11. Thiostreptonresistant transformants of SE180-11 were isolated, the presence oferythromycin resistance was determined, and Thio^(r) Erm^(r)transformants were analyzed by fermentation. The presence of the doublemutated aveC gene, encoding S138T/A139T, was able to restore normalavermectin production to strain SE180-11; however, the B2:B1 ratio was0.84:1 showing that this mutation further reduces the amount ofcyclohexyl-B2 produced relative to cyclohexyl-B1 (TABLE 3), over thereductions provided by transformation of strain SE180-11 with pSE188 orpSE199, as described above.

[0224] Another mutation was constructed to further reduce the amount ofcyclohexyl-B2 produced relative to cyclohexyl-B1. Because theS138T/A139T mutations altered the B2:B1 ratios in the more favorable B1direction, a mutation was constructed to introduce a threonine at aminoacid position 138 and a phenylalanine at amino acid position 139. The˜1.2 Kb insert DNA from pSE186 was used as a PCR template. The PCRprimers were designed to introduce mutations at nt positions 585(changing a T to A), 588 (changing a G to T), and 589 (changing a C toT), and were supplied by Genosys Biotechnologies, Inc. (Tex.). Therightward PCR primer was:5′-GGGGGCGGGCCCGGGTGCGGAGGCGGAAATGCCGCTGGCGACGTTC-3′ (SEQ ID NO;25); andthe leftward PCR primer was: 5′-GGAACATCACGGCATTCACC-3′ (SEQ ID NO:15).The PCR reaction was carried out using an Advantage GC genomic PCR kit(Clonetech Laboratories, Palo Alto, Calif.) in buffer provided by themanufacturer in the presence of 200 μM dNTPs, 200 pmol of each primer,50 ng template DNA, 1.1 mM Mg acetate, 1.0 M GC-Melt and 1 unit Tth DNAPolymerase in a final volume of 50 μl. The thermal profile of the firstcycle was 94° C. for 1 min; followed by 25 cycles of 94° C. for 30 secand 68° C. for 2 min; and 1 cycle at 68° C. for 3 min. A PCR product of449 bp was digested with Apal and Kpnl to release a 254 bp fragment,which was resolved by electrophoresis and purified from the gel. Allthree fragments (˜3.8 Kb, ˜0.4 Kb and 254 bp) were ligated together in a3-way ligation. The ligation mixture was transformed into competent E.coli DH5α cells. Plasmid DNA was isolated from ampicillin resistanttransformants, and the presence of the correct insert was confirmed byrestriction analysis. This plasmid was designated as pSE238.

[0225] pSE238 was digested with EcoRI, cloned into pWHM3, which had beendigested with EcoRI, and transformed into E. coli DH5α cells. PlasmidDNA was isolated from ampicillin resistant transformants and thepresence of the correct insert was confirmed by restriction analysis andDNA sequence analysis. This plasmid DNA was transformed into E. coliDM1, plasmid DNA was isolated from ampicillin resistant transformants,and the presence of the correct insert was confirmed by restrictionanalysis. This plasmid, which was designated as pSE239, was used totransform protoplasts of S. avermitilis strain SE180-11. Thiostreptonresistant transformants of strain SE180-11 were isolated, the presenceof erythromycin resistance was determined, and Thio^(r) Erm^(r)transformants were analyzed by HPLC analysis of fermentation products.The presence of the double mutated aveC gene encoding S138T/A139F wasable to restore normal avermectin production to strain SE180-11;however, the B2:B1 ratio was 0.75:1 showing that this mutation furtherreduced the amount of cyclohexyl-B2 produced relative to cyclohexyl-B1(TABLE 3) over the reductions provided by transformation of strainSE180-11 with pSE188, pSE199, or pSE231 as described above. TABLE 3 No.Avg. S. avermitilis strain Transformants Relative Relative B2:B1(transforming plasmid) Tested B2 Conc. B1 Conc. Ratio SE180-11 (none) 300 0 0 SE180-11 (pWHM3) 30 0 0 0 SE180-11 (pSE186) 26 222 140 1.59SE180-11 (pSE187) 12 283 11 26.3 SE180-11 (pSE188) 24 193 206 0.94SE180-11 (pSE199) 18 155 171 0.88 SE180-11 (pSE231) 6 259 309 0.84SE180-11 (pSE239) 20 184 242 0.75

[0226] Additional mutations were constructed to further reduce theamount of cyclohexyl-B2 produced relative to cyclohexyl-B1 using thetechnique of DNA shuffling as described in Stemmer, 1994, Nature370:389391; and Stemmer, 1994, Proc. Natl. Acad. Sci. USA91:10747-10751; and further described in U.S. Pat. Nos. 5,605,793,5,811,238, 5,830,721, and 5,837,458.

[0227] DNA shuffled plasmids containing mutated aveC genes weretransformed into competent dam dcm E. coli cells. Plasmid DNA wasisolated from ampicillin resistant transformants, and used to transformprotoplasts of S. avermitilis strain SE180-11. Thiostrepton resistanttransformants of strain SE180-11 were isolated and screened for theproduction of avermectins with a cyclohexyl-B2:cyclohexyl-B1 ratio of1:1 or less. The DNA sequence of plasmid DNA from SE180-11 transformantsproducing avermectins with a B2:B1 ratio of 1:1 or less was determined.

[0228] Eight transformants were identified that produced reduced amountsof cyclohexyl-B2 relative to cyclohexyl-B1. The lowest B2:B1 ratioachieved among these transformants was 04:1 (TABLE 4). Plasmid DNA wasisolated from each of the eight transformants and the, DNA sequencedetermined to identify the mutations in the aveC gene. The mutations areas follows.

[0229] pSE290 contains 4 nucleotide mutations at nt position 317 from Tto A, at nt position 353 from C to A, at nt position 438 from G to A,and at nt position 1155 from T to A. The nucleotide change at ntposition 317 changes the amino acid at AA position 48 from D to E andthe nucleotide change at nt position 438 changes the amino acid at AAposition 89 from A to T. The B2:B1 ratio produced by cells carrying thisplasmid was 0.42:1 (TABLE 4). pSE291 contains 4 nucleotide mutations atnt position 272 from G to A, at nt position 585 from T to A, at ntposition 588 from G to A, and at nt position 708 from G to A. Thenucleotide change at nt position 585 changes the amino acid at Mposition 138 from S to T, the nucleotide change at nt position 588changes the amino acid at AA position 139 from A to T, and thenucleotide change at nt position 708 changes the amino acid at AAposition 179 from G to S. The B2:B1 ratio produced by cells carryingthis plasmid was 0.57:1 (TABLE 4).

[0230] pSE292 contains the same four nucleotide mutations as pSE290. TheB2:B1 ratio produced by cells carrying this plasmid was 0.40:1 (TABLE4).

[0231] pSE293 contains 6 nucleotide mutations at nt 24 from A to G, atnt position 286 from A to C, at nt position 497 from T to C, at ntposition 554 from C to T, at nt position 580 from T to C, and at ntposition 886 from A to T. The nucleotide change at nt position 286changes the amino acid at AA position 38 from Q to P, the nucleotidechange at nt position 580 changes the amino acid at AA position 136 fromL to P, and the nucleotide change at nt position 886 changes the aminoacid at AA position 238 from E to D. The B2:B1 ratio produced by cellscarrying this plasmid was 0.68:1 (TABLE4).

[0232] pSE294 contains 6 nucleotide mutations at nt 469 from T to C, atnt position 585 from T to A, at nt position 588 from G to A, at ntposition 708 from G to A, at nt position 833 from C to T, and at ntposition 1184 from G to A. In addition, nts at positions 173, 174, and175 are deleted. The nucleotide change at nt position 469 changes theamino acid at AA position 99 from F to S, the nucleotide change at ntposition 585 changes the amino acid at AA position 138 from S to T, thenucleotide change at nt position 588 changes the amino acid at AAposition 139 from A to T, and the nucleotide change at nt position 708changes the amino acid from AA position 179 from G to S. The B2:B1 ratioproduced by cells carrying this plasmid was 0.53:1 (TABLE 4).

[0233] pSE295 contains 2 nucleotide mutations at nt 588 from G to A andat nt 856 from T to C. The nucleotide change at nt position 588 changesthe amino acid at AA position 139 from A to T and the nucleotide changeat nt position 856 changes the amino acid at AA position 228 from M toT. The B2:B1 ratio produced by cells carrying this plasmid was 0.80:1(TABLE 4).

[0234] pSE296 contains 5 nucleotide mutations at nt position 155 from Tto C, at nt position 505 from G to T, at nt position 1039 from C to T,at nt position 1202 from C to T, and at nt position 1210 from T to C.The nucleotide change at nt position 505 changes the amino acid at PAposition 111 from G to V and the nucleotide change at nt position 1039changes the amino acid at AA position 289 from P to L. The B2:B1 ratioproduced by cells carrying this plasmid was 0.73:1 (TABLE 4).

[0235] pSE297 contains 4 nucleotide mutations at nt position 377 from Gto T, at nt position 588 from G to A, at nt position 633 from A to G,and at nt position 1067 from A to T. The nucleotide change at ntposition 588 changes the amino acid at AA position 139 from A to T, thenucleotide change at nt position 633 changes the amino acid at AAposition 154 from K to E, and the nucleotide change at nt position 1067changes the amino acid at AA position 298 from Q to H. The B2:B1 ratioproduced by cells carrying this plasmid was 0.67:1 (TABLE 4). TABLE 4No. Avg. S. avermitilis strain Transformants Relative Relative B2:B1(transforming plasmid) Tested B2 Conc. B1 Conc. Ratio SE180-11 (none) 40 0 0 SE180-11 (pWHM3) 4 0 0 0 SE180-11 (pSE290) 4 87 208 0.42 SE180-11(pSE291) 4 106 185 0.57 SE180-11 (pSE292) 4 91 231 0.40 SE180-11(pSE293) 4 123 180 0.68 SE180-11 (pSE294) 4 68 129 0.53 SE180-11(pSE295) 4 217 271 0.80 SE180-11 (pSE296) 1 135 186 0.73 SE180-11(pSE297) 1 148 221 0.67

9. EXAMPLE: CONSTRUCTION OF 5′ DELETION MUTANTS

[0236] As explained in Section 5.1, above, the S. avermitilis nucleotidesequence shown in FIG. 1 (SEQ ID NO:1) comprises four different GTGcodons at bp positions 42, 174, 177 and 180 which are potential startsites. This section describes the construction of multiple deletions ofthe 5′ region of the aveC ORF (FIG. 1; SEQ ID NO:1) to help define whichof these codons could function as start sites in the aveC ORF forprotein expression.

[0237] Fragments of the aveC gene variously deleted at the 5′ end wereisolated from S. avermitilis chromosomal DNA by PCR amplification. ThePCR primers were designed based on the aveC DNA sequence, and weresupplied by Genosys Biotechnologies, Inc. The rightward primers were-5′-AACCCATCCGAGCCGCTC3′ (SEQ ID NO:16) (D1F1); 5′-TCGGCCTGCCAACGAAC-3′(SEQ ID NO:17) (D1F2); 5′-CCAACGAACGTGTAGTAG-3′ (SEQ ID NO:18) (D1F3);and 5′-TGCAGGCGTACGTGTTCAGC-3′ (SEQ ID NO:19) (D2F2). The leftwardprimers were 5′-CATGATCGCTGAACCGA-3′ (SEQ ID NO:20);5CATGATCGCTGAACCGAGGA-3′ (SEQ ID NO:21); and 5′-AGGAGTGTGGTGCGTCTGGA-3′(SEQ. ID NO:22). The PCR reaction was carried out as described inSection 8.3, above.

[0238] The PCR products were separated by electrophoresis in a 1%agarose gel and single DNA bands of either ˜1.0 Kb or ˜1.1 Kb weredetected. The PCR products were purified from the gel and ligated with25 ng of linearized pCR2.1 vector (Invitrogen) in a 1:10 molarvector-to-insert ratio following the manufacturer's instructions. Theligation mixtures were used to transform One Shot™ Competent E. colicells (Invitrogen) following manufacturer's instructions. Plasmid DNAwas isolated from ampicillin resistant transformants and the presence ofthe insert was confirmed by restriction analysis and DNA sequenceanalysis. These plasmids were designated as pSE190 (obtained with primerD1F1), pSE191 (obtained with primer D1F2), pSE192 (obtained with primerD1F3), and pSE193 (obtained with primer D2F2).

[0239] The insert DNAs were each digested with BamHI/Xbal, separated byelectrophoresis, purified from the gel, and separately ligated withshuttle vector pWHM3, which had been digested with BamHI/Xbal, in atotal DNA concentration of 1 μg in a 1:5 molar vector-to-insert ratio.The ligation mixtures were used to transform competent E. coli DH5αcells. Plasmid DNA was isolated from ampicillin resistant transformantsand the presence of the insert was confirmed by restriction analysis.These plasmids, which were designated as pSE194 (D1F1), pSE195 (D1F2),pSE196 (D1F3), and pSE197 (D2F2), were each separately transformed intoE. coli strain DM1, plasmid DNA isolated from ampicillin resistanttransformants, and the presence of the correct insert confirmed byrestriction analysis. This DNA was used to transform protoplasts of S.avermitilis strain SE180-11. Thiostrepton resistant transformants ofstrain SE1850-11 were isolated, the presence of erythromycin resistancewas determined, and Thio^(r) Erm^(r) transformants were analyzed by HPLCanalysis of fermentation products to determine which GTG sites werenecessary for avec expression. The results indicate that the GTG codonat position 42 can be eliminated without affecting avec expression,since pSE194, pSE195, and pSE196, each of which lack the GTG site atposition 42, but which all contain the three GTG sites at positions 174,177, and 180, were each able to restore normal avermectin productionwhen transformed into SE180-11. Normal avermectin production was notrestored when strain SE180-11 was transformed with pSE197, which lacksall four of the GTG sites (TABLE 5). TABLE 5 No. Avg. S. avermitilisstrain Transformants Relative Relative B2:B1 (transforming plasmid)tested B2 Conc. B1 Conc. Ratio SE180-11 (none) 6 0 0 0 SE180-11 (pWHM3)6 0 0 0 SE180-11 (pSE186) 6 241 152 1.58 SE180-11 (pSE194) 6 35 15 2.43SE180-11 (pSE195) 6 74 38 1.97 SE180-11 (pSE196) 6 328 208 1.58 SE180-11(pSE197) 12 0 0 0

10. EXAMPLE: CLONING OF aveC HOMOLOGS FROM S. HYGROSCOPICUS AND S.GRISEOCHROMOGENES

[0240] The present invention allows aveC homolog genes from otheravermectin- or milbernycin-producing species of Streptomyces to beidentified and cloned. For example, a cosmid library of S. hygroscopicus(FERM BP-1901) genomic DNA was hybridized with the 1.2 Kb aveC probefrom S. avermitilis described above. Several cosmid clones wereidentified that hybridized strongly. Chromosomal DNA was isolated fromthese cosmids, and a 4.9 Kb Kpnl fragment was identified that hybridizedwith the aveC probe. This DNA was sequenced and an ORF (SEQ ID NO:3) wasidentified having significant homology to the aveC ORF of S.avermitilis. An amino acid sequence (SEQ ID NO:4) deduced from the S.hygroscopicus aveC homolog ORF is presented in FIG. 6.

[0241] In addition, a cosmid library of S. griseochromogenes genomic DNAwas hybridized with the 1.2 Kb aveC probe from S. avermitilis describedabove. Several cosmid clones were identified that hybridized strongly.Chromosomal DNA was isolated from these cosmids, and a 5.4 Kb Pstlfragment was identified that hybridized with the aveC probe. This DNAwas sequenced and an aveC hornolog partial ORF was identified havingsignificant homology to the aveC ORF of S. avermitilis. A deducedpartial amino acid sequence (SEQ ID NO:5) is presented in FIG. 6.

[0242] DNA and amino acid sequence analysis of the aveC homologs from S.hygroscopicus and S. griseochromogenes indicates that these regionsshare significant homology (˜50% sequence identity at the amino acidlevel) both to each other and to the S. avermitilis aveC ORF and AveCgene product (FIG. 6).

11. EXAMPLE: CONSTRUCTION OF A PLASMID WITH THE aveC GENE BEHIND THEermE PROMOTER

[0243] The 1.2 Kb aveC ORF from pSE186 was subcloned in pSE34, which isthe shuttle vector pWHM3 having the 300 bp ermE promoter inserted as aKpnl/BamHI fragment in the Kpnl/BamHI site of pWHM3 (see Ward et al.,1986, Mol. Gen. Genet 203:468 478). pSE186 was digested with BamHI andHindIII, the digest resolved by electrophoresis, and the 1.2 Kb fragmentwas isolated from the agarose gel and ligated with pSE34, which had beendigested with BamHI and HindIII. The ligation mixture was transformedinto competent E. coli DH5α cells according to manufacturer'sinstructions. Plasmid DNA was isolated from ampicillin resistanttransformants, and the presence of the 1.2 Kb insert was confirmed byrestriction analysis. This plasmid, which was designated as pSE189, wastransformed into E. coli DM1, and plasmid DNA isolated from ampicillinresistant transformants. Protoplasts of S. avermitilis strain 1100-SC38were transformed with pSE189. Thiostrepton resistant transformants ofstrain 1100-SC38 were isolated and analyzed by HPLC analysis offermentation products.

[0244]S. avermitilis strain 1100-SC38 transformants containing pSE189were altered in the ratios of avermectin cyclohexyl-B2:avermectincyclohexyl-B1 produced (about 3:1) compared to strain 1100-SC38 (about34:1), and total avermectin production was increased approximately2.4-fold compared to strain 1100-SC38 transformed with pSE119 (TABLE 6).

[0245] pSE189 was also transformed into protoplasts of a wild-type S.avermitilis strain. Thiostrepton resistant transformants were isolatedand analyzed by HPLC analysis of fermentation products. Totalavermectins produced by S. avermitilis wild-type transformed with pSE189were increased approximately 2.2-fold compared to wild-type S.avermitilis transformed with pSE119 (TABLE 6). TABLE 6 S. avermitilisstrain No. Trans- Avg. (transforming formants Relative Relative RelativeTotal B2:B1 plasmid) Tested [B2] [B1] Avermectins Ratio 1100-SC38 6 1554.8 176 33.9 1100-SC38 9 239 50.3 357 4.7 (pSE119) 1100-SC38 16 546 166849 3.3 (pSE189) wild type 6 59 42 113 1.41 wild type 6 248 151 481 1.64(pSE119) wild type 5 545 345 1,071 1.58 (pSE189)

12. EXAMPLE: CHIMERIC PLASMID CONTAINING SEQUENCES FROM BOTH S.AVERMITILIS aveC ORF AND S. HYGROSCOPICUS aveC HOMOLOG

[0246] A hybrid plasmid designated as pSE350 was constructed thatcontains a 564 bp portion of the S. hygroscopicus aveC homolog replacinga 564 bp homologous portion of the S. avermitilis aveC ORF (FIG. 7), asfollows. pSE350 was constructed using a BsaAl restriction site that isconserved in both sequences (aveC position 225), and a Kpnl restrictionsite that is present in the S. avermitilis aveC gene (aveC position810). The Kpnl site was introduced into the S. hygroscopicus DNA by PCRusing the rightward primer 5′-CTTCAGGTGTACGTGTTCG-3′ (SEQ ID NO:23) andthe leftward primer 5′-GAACTGGTACCAGTGCCC-3′ (SEQ ID NO:24) (supplied byGenosys Biotechnologies) using PCR conditions described in Section7.1.10, above. The PCR product was digested with BsaAI and Kpnl, thefragments were separated by electrophoresis in a 1% agarose gel, and the564 bp BsaAl/Kpnl fragment was isolated from the gel. pSE179 (describedin Section 7.1.10, above) was digested with Kpnl and HindIII, thefragments separated by electrophoresis in a 1% agarose gel, and afragment of ˜4.5 Kb was isolated from the gel. pSE179 was digested withHindIII and BsaAI, the fragments separated by electrophoresis in a 1%agarose gel, and a ˜0.2 Kb BsaAI/HindIII fragment isolated from the gel.The ˜4.5 Kb HindIII/Kpnl fragment the ˜0.2 Kb BsaAI/HindIII fragment,and the 564 bp BsaAI/Kpnl fragment from S. hygroscopicus were ligatedtogether in a 3-way ligation and the ligation mixture transformed intocompetent E. coli DH5α cells. Plasmid DNA was isolated from ampicillinresistant transformants and the presence of the correct insert wasconfirmed by restriction analysis using Kpnl and Aval. This plasmid wasdigested with HindIII and Xbal to release the 1.2 Kb insert, which wasthen ligated with pWHM3 which had been digested with HindIII and Xbal.The ligation mixture was transformed into competent E. coli DH5α cells,plasmid DNA was isolated from ampicillin resistant transformants, andthe presence of the correct insert was confirmed by restriction analysisusing HindIII and Aval. This plasmid DNA was transformed into E. coliDM1, plasmid DNA was isolated from ampicillin resistant transformants,and the presence of the correct insert was confirmed by restrictionanalysis and DNA sequence analysis. This plasmid was designated aspSE350 and used to transform protoplasts of S. avermitilis strainSE180-11. Thiostrepton resistant transformants of strain SE180-11 wereisolated, the presence of erythromycin resistance was determined andThio^(r) Erm^(r) transformants were analyzed by HPLC analysis offermentation products. Results show that transformants containing the S.avermitilis/S. hygroscopicus hybrid plasmid have an average B2:B1 ratioof about 109:1 (TABLE 7). TABLE 7 No. Avg. S. avermitilis strainTransformants Relative Relative B2:B1 (transforming plasmid) tested B2Conc. B1 Conc. Ratio SE180-11 (none) 8 0 0 0 SE180-11 (pWHM3) 8 0 0 0SE180-11 (pSE350) 16 233 2 109

DEPOSIT OF BIOLOGICAL MATERIALS

[0247] The following biological material was deposited with the AmericanType Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, Md.,20852, USA, on Jan. 29, 1998, and was assigned the following accessionnumbers: Plasmid Accession No. plasmid pSE180 209605 plasmid pSE186209604

[0248] All patents, patent applications, and publications cited aboveare incorporated herein by reference in their entirety.

[0249] The present invention is not to be limited in scope by thespecific embodiment described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

1 25 1 1229 DNA Streptomyces avermitilis CDS (174)..(1085) 1 tcacgaaaccggacacacca cacacacgaa ggtgagacag cgtgaaccca tccgagccgc 60 tcggcctgcccaacgaacgt gtagtagaca cccgaccgtc cgatgccacg ctctcacccg 120 aggccggcctgaacaggtca ggagcgctgc cccgtgaact gctgtcgttg ccg gtg 176 Val 1 gtg gtgtgg gcc ggg gtc ggc ctg ctg ttt ctg gcc ctg cag gcg tac 224 Val Val TrpAla Gly Val Gly Leu Leu Phe Leu Ala Leu Gln Ala Tyr 5 10 15 gtg ttc agccgc tgg gcg gcc gac ggt ggc tac cgg ctg atc gag acg 272 Val Phe Ser ArgTrp Ala Ala Asp Gly Gly Tyr Arg Leu Ile Glu Thr 20 25 30 gcg ggc cag ggtcag ggc ggc agc aag gat acg ggg act acc gat gtg 320 Ala Gly Gln Gly GlnGly Gly Ser Lys Asp Thr Gly Thr Thr Asp Val 35 40 45 gtc tat ccc gtg atttcc gtc gtc tgc atc acc gcc gcg gcg gcg tgg 368 Val Tyr Pro Val Ile SerVal Val Cys Ile Thr Ala Ala Ala Ala Trp 50 55 60 65 ctc ttc cgg agg tgccgt gtc gaa cga cgg ctg ctg ttc gac gcc ctt 416 Leu Phe Arg Arg Cys ArgVal Glu Arg Arg Leu Leu Phe Asp Ala Leu 70 75 80 ctc ttc ctc ggg ctg ctgttc gcg agc tgg cag agc ccg ctc atg aac 464 Leu Phe Leu Gly Leu Leu PheAla Ser Trp Gln Ser Pro Leu Met Asn 85 90 95 tgg ttc cat tcc gtt ctc gtctcc aac gcg agt gtg tgg ggc gcg gtg 512 Trp Phe His Ser Val Leu Val SerAsn Ala Ser Val Trp Gly Ala Val 100 105 110 ggt tcc tgg ggt ccg tat gtgccc ggc tgg cag ggg gcg ggc ccg ggt 560 Gly Ser Trp Gly Pro Tyr Val ProGly Trp Gln Gly Ala Gly Pro Gly 115 120 125 gcg gag gcg gaa atg ccg ctggcg tcg gcc tcc gtc tgc atg tcg gct 608 Ala Glu Ala Glu Met Pro Leu AlaSer Ala Ser Val Cys Met Ser Ala 130 135 140 145 ctg atc gtc acc gtg ctgtgc agc aag gca ctg ggg tgg atc aag gcc 656 Leu Ile Val Thr Val Leu CysSer Lys Ala Leu Gly Trp Ile Lys Ala 150 155 160 cgc cgg ccg gca tgg cggacc tgg cgg ctg gtc ctg gcc gtg ttc ttc 704 Arg Arg Pro Ala Trp Arg ThrTrp Arg Leu Val Leu Ala Val Phe Phe 165 170 175 atc ggc atc gtg ctc ggtctg tcc gag ccg ctg ccg tcc gcc tcc ggg 752 Ile Gly Ile Val Leu Gly LeuSer Glu Pro Leu Pro Ser Ala Ser Gly 180 185 190 atc agc gta tgg gcc agagcg ctg ccc gag gtg acc ttg tgg agt ggc 800 Ile Ser Val Trp Ala Arg AlaLeu Pro Glu Val Thr Leu Trp Ser Gly 195 200 205 gag tgg tac cag ttc cccgtg tat cag gcg gtc ggt tcc ggc ctg gtc 848 Glu Trp Tyr Gln Phe Pro ValTyr Gln Ala Val Gly Ser Gly Leu Val 210 215 220 225 tgc tgc atg ctg ggctcg ctg cgc ttc ttc cgc gac gaa cgc gat gag 896 Cys Cys Met Leu Gly SerLeu Arg Phe Phe Arg Asp Glu Arg Asp Glu 230 235 240 tcg tgg gtg gaa cgggga gcc tgg cgg ttg ccg caa cgg gca gcg aac 944 Ser Trp Val Glu Arg GlyAla Trp Arg Leu Pro Gln Arg Ala Ala Asn 245 250 255 tgg gcg cgt ttc ctcgcc gtg gtc ggt ggg gtg aat gcc gtg atg ttc 992 Trp Ala Arg Phe Leu AlaVal Val Gly Gly Val Asn Ala Val Met Phe 260 265 270 ctc tac acc tgt ttccat atc ctc ctg tcc ctc gtc ggt gga cag ccg 1040 Leu Tyr Thr Cys Phe HisIle Leu Leu Ser Leu Val Gly Gly Gln Pro 275 280 285 ccc gac caa ctg ccggac tcc ttc caa gcg ccg gcc gct tac tga 1085 Pro Asp Gln Leu Pro Asp SerPhe Gln Ala Pro Ala Ala Tyr 290 295 300 gttcagggca ggtcggagga gacggagaaggggaggcgac cggagttccg gtcacctccc 1145 ctttgtgcat gggtggacgg ggatcacgctcccatggcgg cgggctcctc cagacgcacc 1205 acactcctcg gttcagcgat catg 1229 2303 PRT Streptomyces avermitilis 2 Val Val Val Trp Ala Gly Val Gly LeuLeu Phe Leu Ala Leu Gln Ala 1 5 10 15 Tyr Val Phe Ser Arg Trp Ala AlaAsp Gly Gly Tyr Arg Leu Ile Glu 20 25 30 Thr Ala Gly Gln Gly Gln Gly GlySer Lys Asp Thr Gly Thr Thr Asp 35 40 45 Val Val Tyr Pro Val Ile Ser ValVal Cys Ile Thr Ala Ala Ala Ala 50 55 60 Trp Leu Phe Arg Arg Cys Arg ValGlu Arg Arg Leu Leu Phe Asp Ala 65 70 75 80 Leu Leu Phe Leu Gly Leu LeuPhe Ala Ser Trp Gln Ser Pro Leu Met 85 90 95 Asn Trp Phe His Ser Val LeuVal Ser Asn Ala Ser Val Trp Gly Ala 100 105 110 Val Gly Ser Trp Gly ProTyr Val Pro Gly Trp Gln Gly Ala Gly Pro 115 120 125 Gly Ala Glu Ala GluMet Pro Leu Ala Ser Ala Ser Val Cys Met Ser 130 135 140 Ala Leu Ile ValThr Val Leu Cys Ser Lys Ala Leu Gly Trp Ile Lys 145 150 155 160 Ala ArgArg Pro Ala Trp Arg Thr Trp Arg Leu Val Leu Ala Val Phe 165 170 175 PheIle Gly Ile Val Leu Gly Leu Ser Glu Pro Leu Pro Ser Ala Ser 180 185 190Gly Ile Ser Val Trp Ala Arg Ala Leu Pro Glu Val Thr Leu Trp Ser 195 200205 Gly Glu Trp Tyr Gln Phe Pro Val Tyr Gln Ala Val Gly Ser Gly Leu 210215 220 Val Cys Cys Met Leu Gly Ser Leu Arg Phe Phe Arg Asp Glu Arg Asp225 230 235 240 Glu Ser Trp Val Glu Arg Gly Ala Trp Arg Leu Pro Gln ArgAla Ala 245 250 255 Asn Trp Ala Arg Phe Leu Ala Val Val Gly Gly Val AsnAla Val Met 260 265 270 Phe Leu Tyr Thr Cys Phe His Ile Leu Leu Ser LeuVal Gly Gly Gln 275 280 285 Pro Pro Asp Gln Leu Pro Asp Ser Phe Gln AlaPro Ala Ala Tyr 290 295 300 3 1150 DNA Streptomyces hygroscopicus CDS(58)..(990) 3 gtcgacgaag accggccgga ggccgtcggc cgggccgata ccgtacgcggcctgcgg 57 gtg ttc acc ctt ccc gta aca ctg tgg gcg tgt gtc ggc gcg ctggtg 105 Val Phe Thr Leu Pro Val Thr Leu Trp Ala Cys Val Gly Ala Leu Val1 5 10 15 ctg gga ctt cag gtg tac gtg ttc gcc gcc tgg ctc gcc gac agcggc 153 Leu Gly Leu Gln Val Tyr Val Phe Ala Ala Trp Leu Ala Asp Ser Gly20 25 30 tac cgc atc gag aag gcg tcc ccg gcc agg ggc ggt ggg gac tcg gag201 Tyr Arg Ile Glu Lys Ala Ser Pro Ala Arg Gly Gly Gly Asp Ser Glu 3540 45 cgg atc gcc gat gtg ctg atc ccg ctg ctg tcc gtg gtg gga gcg gtg249 Arg Ile Ala Asp Val Leu Ile Pro Leu Leu Ser Val Val Gly Ala Val 5055 60 gtc ctc gca gtg tgt ctg tac cgg agg tgt cgg gcc agg agg cgg ctg297 Val Leu Ala Val Cys Leu Tyr Arg Arg Cys Arg Ala Arg Arg Arg Leu 6570 75 80 acg ttc gac gcg tcg ctc ttc atc ggg ctg ctg tcg gcc agt tgg cag345 Thr Phe Asp Ala Ser Leu Phe Ile Gly Leu Leu Ser Ala Ser Trp Gln 8590 95 agt ccc ttg atg aac tgg atc aat ccg gtg ctc gcg tca aac gtc aat393 Ser Pro Leu Met Asn Trp Ile Asn Pro Val Leu Ala Ser Asn Val Asn 100105 110 gtg ttc gga gcg gtg gcc tcg tgg ggg ccg tat gtg ccc ggt tgg cag441 Val Phe Gly Ala Val Ala Ser Trp Gly Pro Tyr Val Pro Gly Trp Gln 115120 125 ggg gcg ggg gcg cac cag gag gcc gag ctg ccg ctg gcg acc ctg agc489 Gly Ala Gly Ala His Gln Glu Ala Glu Leu Pro Leu Ala Thr Leu Ser 130135 140 atc tgt atg acg gcc atg atg gcc gcc gtg gcc tgc ggc aag ggc atg537 Ile Cys Met Thr Ala Met Met Ala Ala Val Ala Cys Gly Lys Gly Met 145150 155 160 ggt ctt gcc gcc gcc cgg tgg ccg cgg ctg ggg ccg ctc cgg ctgatc 585 Gly Leu Ala Ala Ala Arg Trp Pro Arg Leu Gly Pro Leu Arg Leu Ile165 170 175 gcg ctc ggc ttt ctg ctc gtc gtg ctc ctc gac atc gcc gag ccgctg 633 Ala Leu Gly Phe Leu Leu Val Val Leu Leu Asp Ile Ala Glu Pro Leu180 185 190 gtg tcc ttc gcg ggc gtc tcc gtg tgg acg cgg gca gtg ccc gagctg 681 Val Ser Phe Ala Gly Val Ser Val Trp Thr Arg Ala Val Pro Glu Leu195 200 205 acc atc tgg agt ggg cac tgg tat cag ttc ccg ctg tat cag atggtg 729 Thr Ile Trp Ser Gly His Trp Tyr Gln Phe Pro Leu Tyr Gln Met Val210 215 220 gct tcg gcg ctc ttc ggc gcc tct ttg ggg gcc gcg cgc cac tttcgc 777 Ala Ser Ala Leu Phe Gly Ala Ser Leu Gly Ala Ala Arg His Phe Arg225 230 235 240 aac cgg cgc ggc gaa acg tgt ctg gag tcc ggg gcg gcc ctccta ccg 825 Asn Arg Arg Gly Glu Thr Cys Leu Glu Ser Gly Ala Ala Leu LeuPro 245 250 255 gag ggc ccg agg cca tgg gtc cgg ctg ctg gcg gtg gtg ggcggg gcc 873 Glu Gly Pro Arg Pro Trp Val Arg Leu Leu Ala Val Val Gly GlyAla 260 265 270 aac atc agc atc gcc ctc tac acc ggc gca cac ggc gca cacatc ctg 921 Asn Ile Ser Ile Ala Leu Tyr Thr Gly Ala His Gly Ala His IleLeu 275 280 285 ttc tcg ctg atg gac ggc gct ccc ccg gac cgg ctc ccc gaattc ttc 969 Phe Ser Leu Met Asp Gly Ala Pro Pro Asp Arg Leu Pro Glu PhePhe 290 295 300 cgt ccg gcg gcc ggc tac tga gaccgccggc accacccacgtacccgatgt 1020 Arg Pro Ala Ala Gly Tyr 305 310 gcgcgatgtg cctgatgcgcctgatgtacc cggggtgtca tcggctcacc tgtggcgcct 1080 catgcggtga gcgctccgcctcgtccttgt tccggctcct gggctccacg accatacgga 1140 gcggccgggg 1150 4 310PRT Streptomyces hygroscopicus 4 Val Phe Thr Leu Pro Val Thr Leu Trp AlaCys Val Gly Ala Leu Val 1 5 10 15 Leu Gly Leu Gln Val Tyr Val Phe AlaAla Trp Leu Ala Asp Ser Gly 20 25 30 Tyr Arg Ile Glu Lys Ala Ser Pro AlaArg Gly Gly Gly Asp Ser Glu 35 40 45 Arg Ile Ala Asp Val Leu Ile Pro LeuLeu Ser Val Val Gly Ala Val 50 55 60 Val Leu Ala Val Cys Leu Tyr Arg ArgCys Arg Ala Arg Arg Arg Leu 65 70 75 80 Thr Phe Asp Ala Ser Leu Phe IleGly Leu Leu Ser Ala Ser Trp Gln 85 90 95 Ser Pro Leu Met Asn Trp Ile AsnPro Val Leu Ala Ser Asn Val Asn 100 105 110 Val Phe Gly Ala Val Ala SerTrp Gly Pro Tyr Val Pro Gly Trp Gln 115 120 125 Gly Ala Gly Ala His GlnGlu Ala Glu Leu Pro Leu Ala Thr Leu Ser 130 135 140 Ile Cys Met Thr AlaMet Met Ala Ala Val Ala Cys Gly Lys Gly Met 145 150 155 160 Gly Leu AlaAla Ala Arg Trp Pro Arg Leu Gly Pro Leu Arg Leu Ile 165 170 175 Ala LeuGly Phe Leu Leu Val Val Leu Leu Asp Ile Ala Glu Pro Leu 180 185 190 ValSer Phe Ala Gly Val Ser Val Trp Thr Arg Ala Val Pro Glu Leu 195 200 205Thr Ile Trp Ser Gly His Trp Tyr Gln Phe Pro Leu Tyr Gln Met Val 210 215220 Ala Ser Ala Leu Phe Gly Ala Ser Leu Gly Ala Ala Arg His Phe Arg 225230 235 240 Asn Arg Arg Gly Glu Thr Cys Leu Glu Ser Gly Ala Ala Leu LeuPro 245 250 255 Glu Gly Pro Arg Pro Trp Val Arg Leu Leu Ala Val Val GlyGly Ala 260 265 270 Asn Ile Ser Ile Ala Leu Tyr Thr Gly Ala His Gly AlaHis Ile Leu 275 280 285 Phe Ser Leu Met Asp Gly Ala Pro Pro Asp Arg LeuPro Glu Phe Phe 290 295 300 Arg Pro Ala Ala Gly Tyr 305 310 5 215 PRTStreptomyces griseochromogenes 5 Val Ile Gly Trp Ala Ala Leu Gly Ala ValPhe Leu Val Leu Gln Val 1 5 10 15 Tyr Val Phe Ala Arg Trp Thr Ala AspGly Gly Tyr His Leu Ala Asp 20 25 30 Val Ser Gly Pro Asp Gly Arg Glu ProGly His Arg Arg Ile Ile Asp 35 40 45 Val Leu Leu Pro Ala Leu Ser Met AlaGly Val Val Gly Leu Ala Phe 50 55 60 Trp Leu Val Arg Arg Trp Arg Ala GluArg Arg Leu Ser Phe Asp Ala 65 70 75 80 Leu Leu Phe Thr Gly Val Leu PheAla Gly Trp Leu Ser Pro Leu Met 85 90 95 Asn Trp Phe His Pro Val Leu MetAla Asn Thr His Val Trp Gly Ala 100 105 110 Val Gly Ser Trp Gly Pro TyrVal Pro Gly Trp Arg Gly Leu Pro Pro 115 120 125 Gly Lys Glu Ala Glu LeuPro Leu Val Thr Phe Ser Leu Gly Ser Thr 130 135 140 Val Leu Leu Gly ValLeu Gly Cys Cys Gln Val Met Ser Arg Val Arg 145 150 155 160 Glu Arg TrpPro Gly Val Arg Pro Trp Gln Leu Val Gly Leu Ala Phe 165 170 175 Leu ThrAla Val Ala Phe Asp Leu Ser Glu Pro Phe Ile Ser Phe Ala 180 185 190 GlyVal Ser Val Trp Ala Arg Ala Leu Pro Thr Val Thr Leu Trp Arg 195 200 205Gly Ala Trp Tyr Arg Ala Arg 210 215 6 18 DNA Streptomyces avermitilis 6tcacgaaacc ggacacac 18 7 18 DNA Streptomyces avermitilis 7 catgatcgctgaaccgag 18 8 20 DNA Streptomyces avermitilis 8 ggttccggat gccgttctcg 209 21 DNA Streptomyces avermitilis 9 aactccggtc gactcccctt c 21 10 19 DNAStreptomyces avermitilis 10 gcaaggatac ggggactac 19 11 18 DNAStreptomyces avermitilis 11 gaaccgaccg cctgatac 18 12 43 DNAStreptomyces avermitilis 12 gggggcgggc ccgggtgcgg aggcggaaat gcccctggcgacg 43 13 20 DNA Streptomyces avermitilis 13 ggaaccgacc gcctgataca 20 1446 DNA Streptomyces avermitilis 14 gggggcgggc ccgggtgcgg aggcggaaatgccgctggcg acgacc 46 15 20 DNA Streptomyces avermitilis 15 ggaacatcacggcattcacc 20 16 18 DNA Streptomyces avermitilis 16 aacccatccg agccgctc18 17 17 DNA Streptomyces avermitilis 17 tcggcctgcc aacgaac 17 18 18 DNAStreptomyces avermitilis 18 ccaacgaacg tgtagtag 18 19 20 DNAStreptomyces avermitilis 19 tgcaggcgta cgtgttcagc 20 20 17 DNAStreptomyces avermitilis 20 catgatcgct gaaccga 17 21 20 DNA Streptomycesavermitilis 21 catgatcgct gaaccgagga 20 22 20 DNA Streptomycesavermitilis 22 aggagtgtgg tgcgtctgga 20 23 19 DNA Streptomycesavermitilis 23 cttcaggtgt acgtgttcg 19 24 18 DNA Streptomycesavermitilis 24 gaactggtac cagtgccc 18 25 46 DNA Streptomyces avermitilis25 gggggcgggc ccgggtgcgg aggcggaaat gccgctggcg acgttc 46

What is claimed is:
 1. An isolated polynucleotide molecule comprisingthe complete aveC ORF of S. avermitilis, which isolated polynucleotidemolecule lacks the next complete ORF that is located downstream from theaveC ORF in situ in the S. avermitilis chromosome.
 2. The isolatedpolynucleotide molecule of claim 1, comprising a nucleotide sequencethat is the same as the S. avermitilis AveC gene product-encodingsequence of plasmid pSE186 (ATCC 209604), or that is the same as thenucleotide sequence of the aveC ORF of FIG. 1 (SEQ ID NO:1).
 3. Theisolated polynucleotide molecule of claim 2, further comprisingnucleotide sequences that naturally flank the aveC gene in situ in S.avermitilis.
 4. An isolated polynucleotide molecule comprising thenucleotide sequence of SEQ ID NO:1, or a degenerate variant thereof. 5.An isolated polynucleotide molecule, comprising a nucleotide sequencethat is homologous to the S. avermitilis AveC gene product-encodingsequence of plasmid pSE186 (ATCC 209604), or to the nucleotide sequenceof the aveC ORF presented in FIG. 1 (SEQ ID NO:1).
 6. An isolatedpolynucleotide molecule consisting of a nucleotide sequence that is asubstantial portion of: (a) a polynucleotide molecule comprising anucleotide sequence that is the same as the S. avermitilis AveC geneproduct-encoding sequence of plasmid pSE186 (ATCC 209604), or that isthe same as the nucleotide sequence of the aveC ORF of FIG. 1 (SEQ IDNO:1); or (b) a polynucleotide molecule comprising a nucleotide sequencethat is homologous to the S. avermitilis AveC gene product-encodingsequence of plasmid pSE186 (ATCC 209604) or to the nucleotide sequenceof the aveC ORF presented in FIG. 1 (SEQ ID NO:1).
 7. An isolatedpolynucleotide molecule comprising the nucleotide sequence of SEQ IDNO:3, or a nucleotide sequence that is homologous to the nucleotidesequence of SEQ ID NO:3.
 8. An isolated polynucleotide moleculeconsisting of a nucleotide sequence that is a substantial portion of thenucleotide sequence of SEQ ID NO:3 or a nucleotide sequence that ishomologous to the nucleotide sequence of SEQ ID NO:3.
 9. Anoligonucleotide molecule that hybridizes under highly stringentconditions to a polynucleotide molecule having the nucleotide sequenceof FIG. 1 (SEQ ID NO:1) or SEQ ID NO:3, or to a polynucleotide moleculehaving a nucleotide sequence that is the complement of the nucleotidesequence of FIG. 1 (SEQ ID NO:1) or SEQ ID NO:3.
 10. The oligonucleotidemolecule of claim 9, which is complementary to the nucleotide sequenceof FIG. 1 (SEQ ID NO:1) or SEQ ID NO:3, or to a polynucleotide moleculehaving a nucleotide sequence that is the complement of the nucleotidesequence of FIG. 1 (SEQ ID NO:1) or SEQ ID NO:3.
 11. A recombinantvector comprising a polynucleotide molecule comprising a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence that is the same as the S. avermitilis AveC geneproduct-encoding sequence of plasmid pSE186 (ATCC 209604), or that isthe same as the nucleotide sequence of the aveC ORF of FIG. 1 (SEQ IDNO:1); (b) a nucleotide sequence that is homologous to the S.avermitilis AveC gene product-encoding sequence of plasmid pSE186 (ATCC209604), or that is homologous to the nucleotide sequence of the aveCORF of FIG. 1 (SEQ ID NO:1); and (c) the nucleotide sequence of SEQ IDNO:3, or a nucleotide sequence that is homologous to the nucleotidesequence of SEQ ID NO:3.
 12. The vector of claim 11, further comprisinga nucleotide sequence encoding one or more regulatory elements inoperative association with the coding sequence of the vector of claim11.
 13. The vector of claim 12, further comprising a nucleotide sequenceencoding a selectable marker.
 14. The vector of claim 13, which isplasmid pSE186 (ATCC 209604).
 15. A host cell comprising the vector ofclaim
 11. 16. The host cell of claim 15, which is Streptomycesavermitilis or Streptomyces hygroscopicus.
 17. An isolated S.avermitilis AveC gene product having an amino acid sequence encoded bythe S. avermitilis AveC gene product-encoding nucleotide sequence ofplasmid pSE186 (ATCC 209604), or encoded by the nucleotide sequence ofSEQ ID NO:1, or having the amino acid sequence of SEQ ID NO:2.
 18. Anisolated S. hygroscopicus AveC-homolog gene product having the aminoacid sequence of SEQ ID NO:4.
 19. A method for producing a recombinantAveC or AveC-homolog gene product, comprising culturing the host cell ofclaim 15 under conditions conducive to the production of the recombinantAveC or AveC-homolog gene product, and recovering the AveC orAveC-homolog gene product from the cell culture.
 20. An isolatedpolynucleotide molecule comprising a nucleotide sequence that isotherwise the same as the S. avermitilis aveC allele or the AveC geneproduct-encoding sequence of plasmid pSE186 (ATCC 209604) or thenucleotide sequence of the aveC ORF of S. avermitilis as presented inFIG. 1 (SEQ ID NO:1), but which nucleotide sequence further comprisesone or more mutations such that cells of S. avermitilis strain ATCC53692 in which the wild-type aveC allele has been inactivated and thatexpress the polynucleotide molecule comprising the mutated nucleotidesequence produce a different ratio or amount of avermectins than areproduced by cells of S. avermitilis strain ATCC 53692 that insteadexpress only the wild-type aveC allele.
 21. An isolated polynucleotidemolecule comprising a nucleotide sequence that is otherwise the same asa degenerate variant of the S. avermitilis aveC allele or the AveC geneproduct-encoding sequence of plasmid pSE186 (ATCC 209604) or thenucleotide sequence of the aveC ORF of S. avermitilis as presented inFIG. 1 (SEQ ID NO:1), but which nucleotide sequence further comprisesone or more mutations such that cells of S. avermitilis strain ATCC53692 in which the wild-type aveC allele has been inactivated and thatexpress the polynucleotide molecule comprising the mutated nucleotidesequence produce a different ratio or amount of avermectins than areproduced by cells of S. avermitilis strain ATCC 53692, that insteadexpress only the wild-type aveC allele.
 22. The polynucleotide moleculeof claim 20, which modulates avermectin production such that cells of S.avermitilis strain ATCC 53692 in which the wild-type aveC allele hasbeen inactivated and that express the polynucleotide molecule produceavermectins in a reduced class 2:1 ratio compared to cells of S.avermitilis strain ATCC 53692 that instead express only the wild typeaveC allele.
 23. The polynucleotide molecule of claim 20, wherein themutation to the S. avermitilis aveC ORF encodes a substitution at one ormore amino acid residues of the AveC gene product corresponding to aminoacid positions in SEQ ID NO:2 selected from the group consisting ofamino acid residues 55, 138, 139, 230, 266 and
 275. 24. Thepolynucleotide molecule of claim 22, wherein the class 2:1 avermectinsare cyclohexyl B2:cyclohexyl B1 avermectins.
 25. The polynucleotidemolecule of claim 24, wherein the reduced ratio of cyclohexylB2:cyclohexyl B1 is a ratio selected from the group consisting of: lessthan 1.6:1; about 0.94:1; about 0.88:1; and about 0.84:1.
 26. Thepolynucleotide molecule of claim 20, wherein the mutation to the S.avermitilis aveC ORF encodes one or more of the following amino acidsubstitutions at one or more positions corresponding to the followingpositions of the AveC gene product of SEQ ID NO:2: (a) at residue 139from alanine to threonine; (b) at residue 138 from serine to threonine;(c) at residue 138 from serine to threonine and at residue 139 fromalanine to threonine; (d) at residue 55 from serine to phenylalanine;(e) at residue 230 from glycine to aspartate; (f) at residue 266 fromglycine to aspartate, and at residue 275 from tyrosine to histidine 27.The polynucleotide molecule of claim 24, wherein the reduced ratio ofcyclohexyl B2:cyclohexyl B1 is a ratio of about 0.75:1.
 28. Thepolynucleotide molecule of claim 20, wherein the mutation to the S.avermitilis aveC ORF encodes amino acid substitutions at residue 138from serine to, threonine and at residue 139 from alanine tophenylalanine, corresponding to the amino acid positions of the AveCgene product of SEQ ID NO:2.
 29. The polynucleotide molecule of claim20, comprising an aveC allele that has been inactivated by insertioninto said nucleotide sequence of a heterologous nucleotide sequence. 30.The polynucleotide molecule of claim 20, comprising an aveC allele thathas been inactivated by deleting a 640 bp Pstl/Sphl fragment from theaveC ORF as shown in FIG. 1 (SEQ ID NO:1).
 31. The polynucleotidemolecule of claim 20, comprising an aveC allele that has beeninactivated by introducing a frameshift mutation therein.
 32. Thepolynucleotide molecule of claim 20, comprising an aveC allele that hasbeen inactivated by introducing a termination codon into the ORF. 33.The polynucleotide molecule of claim 20 comprising a strong promoter inoperative association with the S. avermitilis aveC allele.
 34. Thepolynucleotide molecule of claim 33, wherein the strong promoter is theermE promoter from Saccharopolyspora erythraea.
 35. A gene replacementvector comprising a polynucleotide molecule comprising one or morenucleotide sequences which flank the aveC ORF in situ in the S.avermitilis chromosome.
 36. A recombinant vector comprising thepolynucleotide molecule of
 20. 37. The recombinant vector of claim 36,which vector is pSE180 (ATCC 209605).
 38. A host Streptomyces cellcomprising the recombinant vector of claim
 37. 39. A method foridentifying mutations of the avec ORF capable of altering the class 2:1ratio of avermectins produced by cells of S. avermitilis, comprising:(a) determining the class 2:1 ratio of avermectins produced by cells ofa strain of S. avermitilis in which the aveC allele native thereto hasbeen inactivated and into which a polynucleotide molecule comprising anucleotide sequence encoding a mutated AveC gene product has beenintroduced and is being expressed; (b) determining the class 2:1 ratioof avermectins produced by cells of the same strain of S. avermitilis asin step (a) but which instead express only a wild-type aveC allele or anucleotide sequence that is homologous thereto; and (c) comparing theclass 2:1 ratio of avermectins produced by the S. avermitilis cells ofstep (a) to the class 2:1 ratio of avermectins produced by the S.avermitilis cells of step (b); such that if the class 2:1 ratio ofavermectins produced by the S. avermitilis cells of step (a) isdifferent from the class 2:1 ratio of avermectins produced by the S.avermitilis cells of step (b), then a mutation of the aveC ORF capableof altering the class 2:1 ratio of avermectins has been identified. 40.The method of claim 39, wherein the class 2:1 ratio of avermectins isreduced by the mutation.
 41. A method for identifying mutations of theaveC ORF or genetic constructs comprising the aveC ORF capable ofaltering the amount of avermectins produced by cells of S. avermitilis,comprising: (a) determining the amount of avermectins produced by cellsof a strain of S. avermitilis in which the aveC allele native theretohas been inactivated and into which a polynucleotide molecule comprisinga nucleotide sequence encoding a mutated AveC gene product or comprisinga genetic construct comprising a nucleotide sequence encoding an AveCgene product has been introduced and is being expressed; (b) determiningthe amount of avermectins produced by cells of the same strain of S.avermitilis as in step (a) but which instead express only a wild-typeaveC allele or a nucleotide sequence that is homologous thereto; and (c)comparing the amount of avermectins produced by the S. avermitilis cellsof step (a) to the amount of avermectins produced by the S. avermitiliscells of step (b); such that if the amount of avermectins produced bythe S. avermitilis cells of step (a) is different from the amount ofavermectins produced by the S. avermitilis cells of step (b), then amutation of the aveC ORF or a genetic construct capable of altering theamount of avermectins has been identified.
 42. The method of claim 41,wherein the amount of avermectins produced is increased by the mutation.43. A method for making novel strains of S. avermitilis comprising cellsthat express a mutated aveC allele and that produce an altered class 2:1ratio of avermectins compared to cells of the same strain of S.avermitilis that instead express only a wild-type aveC allele,comprising: (a) transforming cells of a strain of S. avermitilis with apolynucleotide molecule that carries a mutated aveC allele that encodesa gene product that alters the class 2:1 ratio of avermectins producedby cells of a strain of S. avermitilis expressing the mutated aveCallele compared to cells of the same strain that instead express onlythe wild-type aveC allele, and selecting transformed cells that produceavermectins in an altered class 2:1 ratio compared to the class 2:1ratio produced by cells of the strain that instead express only thewild-type aveC allele; or (b) transforming cells of a strain of S.avermitilis with a polynucleotide molecule capable of introducing amutation into the aveC allele of such cells, wherein the mutated aveCallele encodes a gene product that alters the class 2:1 ratio ofavermectins produced by cells of a strain of S. avermitilis expressingthe mutated aveC allele compared to cells of the same strain thatinstead express only the wild-type aveC allele, and selectingtransformed cells that produce avermectins in an altered class 2:1 ratiocompared to the class 2:1 ratio produced by cells of the strain thatinstead express only the wild-type aveC allele.
 44. The method of claim43, in which cells of the novel strain produce avermectins in a reducedclass 2:1 ratio compared to cells of the same S. avermitilis strain thatinstead express only the wild-type aveC allele.
 45. The method of claim43, wherein the class 2:1 avermectins are cyclohexyl B2:cyclohexyl B1avermectins.
 46. The method of claim 45, wherein the reduced ratio ofcyclohexyl B2:cyclohexyl B1 is a ratio selected from the groupconsisting of: less than 1.6:1; about 0.94:1; about 0.88:1; and about0.84:1.
 47. The method of claim 43, wherein the mutation to the S.avermitilis avec ORF encodes an amino acid substitution at one or morepositions corresponding to the following positions of the AveC geneproduct of SEQ ID NO:2: residue 139; residue 138; residue 55; residue230; residue 266; or residue
 275. 48. The method of claim 43, whereinthe mutation to the S. avermitilis aveC ORF encodes one or more of thefollowing amino acid substitutions at one or more positionscorresponding to the following positions of the AveC gene product of SEQID NO:2: (a) at residue 139 from alanine to threonine; (b) at residue138 from serine to threonine; (c) at residue 138 from serine tothreonine and at residue 139 from alanine to threonine; (d) at residue55 from serine to phenylalanine; (e) at residue 230 from glycine toaspartate; or (f) at residue 266 from glycine to aspartate, and atresidue 275 from tyrosine to histidine
 49. The method of claim 45,wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is about0.75:1.
 50. The method of claim 43, wherein the mutation to the S.avermitilis aveC ORF encodes amino acid substitutions at residue 138from serine to threonine and at residue 139 from alanine tophenylalanine, corresponding to the amino acid positions of the AveCgene product of SEQ ID NO:2.
 51. A strain of Streptomyces avermitiliscomprising cells which comprise a mutated avec allele, which cellsproduce a different ratio or amount of avermectins than that produced bycells of the same S. avermitilis strain which only comprise thewild-type avec allele.
 52. The strain of claim 51, the cells of whichproduce a reduced class 2:1 ratio of avermectins compared to cells ofthe same strain that instead comprise only the wild-type aveC alele. 53.The strain of claim 51, wherein the mutated aveC allele encodes asubstitution at one or more amino acid residues of the AveC gene productcorresponding to amino acid positions in SEQ ID NO:2 selected from thegroup consisting of amino acid residues 55, 138, 139, 230, 266 and 275.54. The strain of claim 52, wherein the class 2:1 avermectins arecyclohexyl B2:cyclohexyl B1 avermectins.
 55. The strain of claim 54,wherein the reduced ratio of cyclohexyl B2:cyclohexyl B1 is a ratioselected from the group consisting of less than 1.6:1; about 0.94:1;about 0.88:1; and about 0.84:1.
 56. The strain of claim 51, wherein themutation to the S. avermitilis aveC ORF encodes one or more of thefollowing amino acid substitutions at one or more positionscorresponding to the following positions of the AveC gene product of SEQID NO:2: (a) at residue 139 from alanine to threonine; (b) at residue138 from serine to threonine; (c) at residue 138 from serine tothreonine and at residue 139 from alanine to threonine; (d) at residue55 from serine to phenylalanine; (e) at residue 230 from glycine toaspartate; or at residue 266 from glycine to aspartate, and at residue275 from tyrosine to histidine.
 57. The strain of claim 54, wherein thereduced ratio of cyclohexyl B2:cyclohexyl B1 is about 0.75:1.
 58. Thestrain of claim 51, wherein the mutation to the S. avermitilis aveC ORFencodes amino acid substitutions at residue 138 from serine to threonineand at residue 139 from alanine to phenylalanine, corresponding to theamino acid positions of the AveC gene product of SEQ ID NO:2.
 59. Thestrain of claim 51, the cells of which comprise an aveC allele that hasbeen inactivated by insertion into said nucleotide sequence of aheterologous nucleotide sequence.
 60. The strain of claim 51, the cellsof which comprise an aveC allele that has been inactivated by deleting a640 bp Pstl/Sphl fragment from the aveC ORF of FIG. 1 (SEQ ID NO:1). 61.The strain of claim 51, the cells of which comprise an aveC allele thathas been inactivated by introducing therein a frameshift mutation. 62.The strain of claim 51, the cells of which comprise an aveC allele thathas been inactivated by introducing a termination codon within the ORF.63. The strain of claim 51, the cells of which comprise a strongpromoter in operative association with the S. avermitilis aveC ORF. 64.The strain of claim 63, wherein the strong promoter is the ermE promoterfrom Saccharopolyspora eryraea.
 65. A process for producing avermectins,comprising culturing cells of a strain of S. avermitilis, which cellsexpress a mutated aveC allele that encodes a gene product that altersthe class 2:1 ratio of avermectins produced by cells of a strain of S.avermitilis expressing the mutated aveC allele compared to cells of thesame strain which do not express the mutated aveC allele but insteadexpress only the wild-type aveC allele, in culture media underconditions that permit or induce the production of avermectinstherefrom, and recovering said avermectins from the culture.
 66. Theprocess of claim 65, wherein the class 2:1 ratio is reduced by themutation.
 67. A process for producing avermectins, comprising culturingcells of a strain of S. avermitilis, which cells express a mutated aveCallele or a genetic construct comprising an aveC allele that results inthe production of an altered amount of avermectins produced by cells ofa strain of S. avermitilis expressing the mutated aveC allele or geneticconstruct compared to cells of the same strain which do not express themutated aveC allele or genetic construct but instead express only awild-type aveC allele, in culture media under conditions that permit orinduce the production of avermectins therefrom, and recovering saidavermectins from the culture.
 68. The process of claim 67, wherein theamount of avermectins produced is increased by expression of the mutatedaveC allele or genetic construct.
 69. A composition of avermectinsproduced by cells of a strain of S. avermitilis, which cells express amutated aveC allele that encodes a gene product that reduces the class2:1 ratio of avermectins produced by the cells compared to cells of thesame strain which do not express the mutated aveC allele but insteadexpress only the wild-type aveC allele, wherein the avermectins areproduced in such a reduced class 2:1 ratio.